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Sample records for cell immobilization electrophoresis

  1. Kidney Cell Electrophoresis

    Science.gov (United States)

    Todd, P.

    1985-01-01

    Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated, ground support in the form of analytical cell electrophoresis and flow cytometry was provided and cells returned from space flight were analyzed. Preflight culture media, electrophoresis buffer, fraction collection media, temperature profiles, and urokinase assay procedures were tested prior to flight. Electrophoretic mobility distributions of aliquots of the cell population to be fractionated in flight were obtained. The protocol established and utilized is given.

  2. Mapping and identification of HeLa cell proteins separated by immobilized pH-gradient two-dimensional gel electrophoresis and construction of a two-dimensional polyacrylamide gel electrophoresis database

    DEFF Research Database (Denmark)

    Shaw, AC; Rossel Larsen, M; Roepstorff, P

    1999-01-01

    The HeLa cell line, a human adenocarcinoma, is used in many research fields, since it can be infected with a wide range of viruses and intracellular bacteria. Therefore, the mapping of HeLa cell proteins is useful for the investigation of parasite host cell interactions. Because of the recent...... improvements of two-dimensional gel electrophoresis with immobilized pH gradients (IPG) compared to isoelectric focusing with carrier ampholytes, a highly reproducible method for examining global changes in HeLa cell protein expression due to different stimuli is now available. Therefore, we have initiated...... the mapping of [35S]methionine/cysteine-labeled HeLa cell proteins with the 2-D PAGE (IPG)-system, using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and N-terminal sequencing for protein identification. To date 21 proteins have been identified and mapped. In order to make...

  3. Improved Electrophoresis Cell

    Science.gov (United States)

    Rhodes, P. H.; Snyder, R. S.

    1982-01-01

    Several proposed modifications are expected to improve performance of a continous-flow electrophoresis cell. Changes would allow better control of buffer flow and would increase resolution by suppressing thermal gradients. Improved electrophoresis device would have high resolution and be easy to operate. Improvements would allow better flow control and heat dissipation.

  4. Immobilized Cell Research

    Science.gov (United States)

    1990-10-31

    beads, the plasmid is twice as stable as in cells In a process where immobilized cells produce material grown in continuous culture over 200...carrageenan) or chemically cross-linked, or- Penicillium chrysogenum than in washed freely suspended ganic polymer (Ca-alginate, polyacrylamide, and mycelium ...these materials are formed into the freely suspended cells stopped after 6 days. If the beads of several millimeters in diameter by allowing the

  5. Down-regulation of triose phosphate isomerase in Vineristine-resistant gastric cancer SGC7901 cell line identified by immobilized pH gradient two-dimensional gel electrophoresis and mierosequencing

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective:To exkplore new multidrug-resistance-related proteins in gastric SC7901 cells and clarify their mechanisms.Methods:Two-dimensional(2-D) polyacrylamide gel electrophoresis with immobilized pH gradients(IPG) was applied to compare the differential expression of multidrug-resistance-related proteins in gastric cancer SGC7901 cells and Vineristine-resistant SGC7901 cells (SGC7901/VCR) induced by vincristine sulfate.The 2-D gels were silver-stained.Then,preparative 2-D PAGE was performed.The differential proteins of PVDF membranes were cxcised and identified by N-terminal microsequencing.The mRNA expressions of differential proteins were detected in SGC 7901 cells and SGC7901/VCR cells by RT-PCR.Results:Approximatedly 680 protein sports were resolved on each 2-D gel by silver staining.Most protein spots showed no difference in composition,shape or density.25 proteins differed in abundance (6 higher in SGC7901/VCR cells;19 higher in 7901 cells);5 proteins were unique to one kind of cell or the othe(3 in SGC7901/VRC cells,2 in 7901 cells).One drug-resistance-related protein,which was down-regulated in SGC7901/VCR cells,was identified as trisephosphate isomerase(TPI),a glycolytic pathway enzyme.Conclusions:the results suggest that these differential proteins including TPI may be related to the Vincristine-resistant mechanism in human gastric cancer SGC7901/VCR cell line.

  6. Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Shaw, AC; Rossel Larsen, M; Roepstorff, P

    1999-01-01

    . A semiconfluent layer of HeLa cells was grown on tissue culture plates, and changes in protein expression due to 100 U/mL IFN-gamma were investigated at different periods after treatment, using pulse labeling with [35S]methionine/cysteine in combination with 2-D PAGE (IPG). The identity of eight protein spots...... was elucidated by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), and several variants of the IFN-gamma-inducible tryptophanyl-tRNA synthetase (hWRS) were detected by immunoblotting....

  7. Engineering aspects of nitrification with immobilized cells.

    NARCIS (Netherlands)

    Hunik, J.H.

    1993-01-01

    Several aspects of a nitrification process with artificially immobilized cells in an airlift loop reactor have been investigated and are described in this thesis. In chapter 1 an overview of immobilization methods, suitable reactors, modelling, small-scaleapplications and scale-up strategy is given.

  8. Simultaneous immobilization of dehydrogenases on polyvinylidene difluoride resin after separation by non-denaturing two-dimensional electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Shimazaki, Youji [Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Bunkyo-cho 2-5, Matsuyama City 790-8577 (Japan)], E-mail: yoji@dpc.ehime-u.ac.jp; Kadota, Mariko [Faculty of Science, Ehime University, Matsuyama (Japan)

    2008-06-16

    We detected mouse liver malate, sorbitol and aldehyde dehydrogenases by negative staining, analysis of malate and sorbitol dehydrogenase activities using each substrate, and electron transfers including nicotinamide adenine dinucleotide (NAD) and nitroblue tetrazolium in non-denaturing two-dimensional electrophoresis (2-DE) gel. Dehydrogenases were also identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) after 2-DE separation and protein detection by negative staining. Spots of dehydrogenases separated by 2-DE were excised, and simultaneously transferred and immobilized on polyvinylidene difuoride (PVDF) resin by electrophoresis. The dehydrogenase activities remained intact after immobilization. In conclusion, resin-immobilized dehydrogenases can be simultaneously obtained after separation by non-denaturing 2-DE, detection by negative staining and transferring to resins.

  9. Surface cell immobilization within perfluoroalkoxy microchannels

    Energy Technology Data Exchange (ETDEWEB)

    Stojkovič, Gorazd; Krivec, Matic [Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana (Slovenia); Vesel, Alenka [Jožef Stefan Institute, Jamova cesta 39, 1000 Ljubljana (Slovenia); Marinšek, Marjan [Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana (Slovenia); Žnidaršič-Plazl, Polona, E-mail: polona.znidarsic@fkkt.uni-lj.si [Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana (Slovenia)

    2014-11-30

    Graphical abstract: - Highlights: • A very efficient approach for immobilization of cells into microreactors is presented. • It is applicable to various materials, including PFA and cyclic olefin (co)polymers. • It was used to immobilize different prokaryotic and eukaryotic microbes. • Cells were immobilized on the surface in high density and showed good stability. • Mechanisms of APTES interactions with target materials are proposed. - Abstract: Perfluoroalkoxy (PFA) is one of the most promising materials for the fabrication of cheap, solvent resistant and reusable microfluidic chips, which have been recently recognized as effective tools for biocatalytic process development. The application of biocatalysts significantly depends on efficient immobilization of enzymes or cells within the reactor enabling long-term biocatalyst use. Functionalization of PFA microchannels by 3-aminopropyltriethoxysilane (ATPES) and glutaraldehyde was used for rapid preparation of microbioreactors with surface-immobilized cells. X-ray photoelectron spectroscopy and scanning electron microscopy were used to accurately monitor individual treatment steps and to select conditions for cell immobilization. The optimized protocol for Saccharomyces cerevisiae immobilization on PFA microchannel walls comprised ethanol surface pretreatment, 4 h contacting with 10% APTES aqueous solution, 10 min treatment with 1% glutaraldehyde and 20 min contacting with cells in deionized water. The same protocol enabled also immobilization of Escherichia coli, Pseudomonas putida and Bacillus subtilis cells on PFA surface in high densities. Furthermore, the developed procedure has been proved to be very efficient also for surface immobilization of tested cells on other materials that are used for microreactor fabrication, including glass, polystyrene, poly (methyl methacrylate), polycarbonate, and two olefin-based polymers, namely Zeonor{sup ®} and Topas{sup ®}.

  10. Immobilization of whole cells using polymeric coatings

    Energy Technology Data Exchange (ETDEWEB)

    Lawton, C.W.; Klei, H.E.; Sunstrom, D.V.; Voronka, P.J.; Scott, C.D. (ed.)

    1986-01-01

    A cell immobilization procedure was developed using latex coatings on solid particles. The method's widespread applicability has been demonstrated by successfully immobilizing Saccharomyces cerevisiae (ethanol production), Bacillus subtilis (tryptophan production). Penicillium chrysogenum (penicillin G production), and Escherichia coli (aspartic acid production). In contrast to other immobilization methods, this procedure produces a pellicular particle that is porous, allowing rapid substrate and gas transfer, has a hard core to avoid compression in large beds, and is dense to allow use in fluidized beds. The immobilization procedure was optimized with S. cerevisiae. Kinetic constants obtained were used to calculate effectiveness factors to show that there was minimal intraparticle diffusion resistance. Reactors utilizing the optimized particles were run for 300 hours to evaluate immobilized particle half-life which was 250 hours.

  11. Surface cell immobilization within perfluoroalkoxy microchannels

    Science.gov (United States)

    Stojkovič, Gorazd; Krivec, Matic; Vesel, Alenka; Marinšek, Marjan; Žnidaršič-Plazl, Polona

    2014-11-01

    Perfluoroalkoxy (PFA) is one of the most promising materials for the fabrication of cheap, solvent resistant and reusable microfluidic chips, which have been recently recognized as effective tools for biocatalytic process development. The application of biocatalysts significantly depends on efficient immobilization of enzymes or cells within the reactor enabling long-term biocatalyst use. Functionalization of PFA microchannels by 3-aminopropyltriethoxysilane (ATPES) and glutaraldehyde was used for rapid preparation of microbioreactors with surface-immobilized cells. X-ray photoelectron spectroscopy and scanning electron microscopy were used to accurately monitor individual treatment steps and to select conditions for cell immobilization. The optimized protocol for Saccharomyces cerevisiae immobilization on PFA microchannel walls comprised ethanol surface pretreatment, 4 h contacting with 10% APTES aqueous solution, 10 min treatment with 1% glutaraldehyde and 20 min contacting with cells in deionized water. The same protocol enabled also immobilization of Escherichia coli, Pseudomonas putida and Bacillus subtilis cells on PFA surface in high densities. Furthermore, the developed procedure has been proved to be very efficient also for surface immobilization of tested cells on other materials that are used for microreactor fabrication, including glass, polystyrene, poly (methyl methacrylate), polycarbonate, and two olefin-based polymers, namely Zeonor® and Topas®.

  12. Capillary electrophoresis-based immobilized enzyme reactor using particle-packing technique.

    Science.gov (United States)

    Liu, Lina; Zhang, Bo; Zhang, Qian; Shi, Yanhong; Guo, Liping; Yang, Li

    2014-07-25

    A novel method using particle-packing technique to fabricate capillary electrophoresis (CE)-based immobilized enzyme reactor (IMER) was accomplished by utilizing perfusive silica single particles as the frits and large-pore beads as the enzyme supports. The fabrication procedure is rapid and simple; the length and enzyme loading amount of the CE-IMERs could be easily adjusted. Performance and feasibility of the CE-IMERs were investigated using on-line trypsin digestion as the model enzyme reaction. High reproducible on-line enzyme assay was demonstrated with RSD less than 4.1% and 3.8% for peak area and migration time of the substrate and product over 100 consecutive runs, respectively. The enzyme can still maintain the activity for at least 10 days, indicating remarkably stability of the CE-IMERs. The CE-IMERs were successfully applied for accurate analysis of trypsin inhibition as well as on-line digestion of standard proteins (myoglobin and BSA). The present method provides a new interesting alternative to open-tubular and monolithic CE-IMERs, thus expands the application of the CE technique for on-line enzyme assay and analysis and characterization of peptides and proteins.

  13. Ethanol tolerance of immobilized brewers' yeast cells.

    Science.gov (United States)

    Norton, S; Watson, K; D'Amore, T

    1995-04-01

    A method based on the survival of yeast cells subjected to an ethanol or heat shock was utilized to compare the stress resistance of free and carrageenan-immobilized yeast cells. Results demonstrated a significant increase of yeast survival against ethanol for immobilized cells as compared to free cells, while no marked difference in heat resistance was observed. When entrapped cells were released by mechanical disruption of the gel beads and submitted to the same ethanol stress, they exhibited a lower survival rate than entrapped cells, but a similar or slightly higher survival rate than free cells. The incidence of ethanol- or heat-induced respiratory-deficient mutants of entrapped cells was equivalent to that of control or non-stressed cells (1.3 +/- 0.5%) whereas ethanol- and heat-shocked free and released cells exhibited between 4.4% and 10.9% average incidence of respiration-deficient mutants. It was concluded that the carrageenan gel matrix provided a protection against ethanol, and that entrapped cells returned to normal physiological behaviour as soon as they were released. The cell growth rate was a significant factor in the resistance of yeast to high ethanol concentrations. The optimum conditions to obtain reliable and reproducible results involved the use of slow-growing cells after exhaustion of the sugar substrate.

  14. Aroma formation by immobilized yeast cells in fermentation processes.

    Science.gov (United States)

    Nedović, V; Gibson, B; Mantzouridou, T F; Bugarski, B; Djordjević, V; Kalušević, A; Paraskevopoulou, A; Sandell, M; Šmogrovičová, D; Yilmaztekin, M

    2015-01-01

    Immobilized cell technology has shown a significant promotional effect on the fermentation of alcoholic beverages such as beer, wine and cider. However, genetic, morphological and physiological alterations occurring in immobilized yeast cells impact on aroma formation during fermentation processes. The focus of this review is exploitation of existing knowledge on the biochemistry and the biological role of flavour production in yeast for the biotechnological production of aroma compounds of industrial importance, by means of immobilized yeast. Various types of carrier materials and immobilization methods proposed for application in beer, wine, fruit wine, cider and mead production are presented. Engineering aspects with special emphasis on immobilized cell bioreactor design, operation and scale-up potential are also discussed. Ultimately, examples of products with improved quality properties within the alcoholic beverages are addressed, together with identification and description of the future perspectives and scope for cell immobilization in fermentation processes.

  15. ENHANCED DEGRADATION OF CAPTAN BY IMMOBILIZED CELLS OF BACILLUS CIRCULANS

    Directory of Open Access Journals (Sweden)

    Veena More

    2014-10-01

    Full Text Available The possibility of using Bacillus circulans in degrading captan was evaluated by comparing the captan degradation rate by freely suspended and immobilized cells on agar, sodium alginate (SA, polyacrylamide (PA and polyurethane-foam (PUF in batch and repeated batch degradations. Under batch degradations, 50, 60, 72, and 88% of 0.1% captan was degraded by freely suspended cells, agar-, SA-, and PA-immobilized cells, respectively in 72 h; whereas 15, 47.5, 67.7 and 75% of 0.2% captan was degraded by freely suspended cells, agar-, SA-, and PA-immobilized cells, respectively in 72 h. However, 0.1 and 0.2% captan were completely degraded by PUF-immobilized cells in 48 and 72 h, respectively. Under repeated batch degradations, PUF-immobilized cells were reused more than 40 cycles for 72 h without losing the captan degradation ability, while the cells immobilized on agar, SA, and the PA could be reused for 15, 20, and 25 cycles, respectively. A significant 0.1% captan degradation by PUF-immobilized cells was observed at pH 4.0 - 10.0 and 20 - 40 ºC ranges. In contrast, freely suspended cells only degraded captan at optimum pH of 7.0 and 30 ºC. The PUF-immobilized cells were able to significantly degrade captan for 120 days at 4 ºC without losing the captan degradation ability; whereas this ability was lost in 120 days for freely suspended cells. Since the application of captan leads to pollution and reduces soil fertility, the use of immobilized cells of Bacillus circulans can thus be a better cost-effective strategy to decontaminate captan polluted sites.

  16. Application of immobilized cells to the treatment of cyanide wastewater.

    Science.gov (United States)

    Chen, C Y; Kao, C M; Chen, S C; Chien, H Y; Lin, C E

    2007-01-01

    Cyanide is highly toxic to living organisms, particularly in inactivating the respiration system by tightly binding to terminal oxidase. To protect the environment and water bodies, wastewater containing cyanide must be treated before discharging into the environment. Biological treatment is a cost-effective and environmentally acceptable method for cyanide removal compared with the other techniques currently in use. Klebsiella oxytoca (K. oxytoca), isolated from cyanide-containing industrial wastewater, has been shown to be able to biodegrade cyanide to non-toxic end products. The technology of immobilized cells can be applied in biological treatment to enhance the efficiency and effectiveness of biodegradation. In this study, potassium cyanide (KCN) was used as the target compound and both alginate (AL) and cellulose triacetate (CTA) techniques were applied for the preparation of immobilized cells. Results from this study show that KCN can be utilized as the sole nitrogen source by K. oxytoca. The free suspension systems reveal that the cell viability was highly affected by initial KCN concentration, pH, and temperature. Results show that immobilized cell systems could tolerate a higher level of KCN concentration and wider ranges of pH and temperature, especially in the system with CTA gel beads. Results show that a longer incubation period was required for KCN degradation using immobilized cells compared to the free suspended systems. This might be due to internal mass transfer limitations. Results also indicate that immobilized systems can support a higher biomass concentration. Complete KCN degradation was observed after the operation of four consecutive degradation experiments with the same batch of immobilized cells. This suggests that the activity of the immobilized cells can be maintained and KCN can be used as the nitrogen source throughout KCN degradation experiments. Results reveal that the application of immobilized cells of K. oxytoca is advantageous

  17. Two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt): the state of the art and the controversy of vertical versus horizontal systems.

    Science.gov (United States)

    Görg, A; Boguth, G; Obermaier, C; Posch, A; Weiss, W

    1995-07-01

    After having established the basic protocol of two-dimensional electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt) in 1988 (A. Görg et al., Electrophoresis 1988, 9, 531-546), some critical parameters of the actual IPG-Dalt protocols as well as the results obtained with horizontal and vertical second-dimensional sodium dodecyl sulfate-electrophoresis are demonstrated and discussed.

  18. Cell immobilization for microbial production of 1,3-propanediol.

    Science.gov (United States)

    Gungormusler-Yilmaz, Mine; Cicek, Nazim; Levin, David B; Azbar, Nuri

    2016-01-01

    Cell and enzyme immobilization are often used for industrial production of high-value products. In recent years, immobilization techniques have been applied to the production of value-added chemicals such as 1,3-Propanediol (1,3-PDO). Biotechnological fermentation is an attractive alternative to current 1,3-PDO production methods, which are primarily thermochemical processes, as it generates high volumetric yields of 1,3-PDO, is a much less energy intensive process, and generates lower amounts of environmental organic pollutants. Although several approaches including: batch, fed-batch, continuous-feed and two-step continuous-feed were tested in suspended systems, it has been well demonstrated that cell immobilization techniques can significantly enhance 1,3-PDO production and allow robust continuous production in smaller bioreactors. This review covers various immobilization methods and their application for 1,3-PDO production.

  19. Ethanol fermentation by immobilized cells of Zymomonas mobilis

    Energy Technology Data Exchange (ETDEWEB)

    Grote, W.

    1985-01-01

    Previous studies have shown that immobilized yeast cell cultures have commercial potential for fuel ethanol production. In this study the suitability of strains of Z. mobilis for whole cell immobilization was investigated. Experiments revealed that immobilization in Ca-alginate or K-carrageenan gel or use of flocculating strains was effective for ethanol production at relatively high productivities. Two laboratory size reactors were designed and constructed. These were a compartmented multiple discshaft column and a tower fermentor. Results of this work supported other studies that established that growth and fermentation could be uncoupled. The data indicated that specific metabolic rates were dependent on the nature of the fermentation media. The addition of lactobacilli to Z. mobilis continuous fermentations had only a transient effect, and was unlikely to affect an immobilized Z. mobilis process. With 150 gl/sup -1/ glucose media and a Z. mobilis ZM4 immobilized cell reactor, a maximum volumetric ethanol productivity of 55 gl/sup -1/h/sup -1/ was obtained. The fermentation of sucrose media or sucrose-based raw materials (molasses, cane juice, synthetic mill liquor) by immobilized Z. mobilis ZM4 revealed a pattern of rapid sucrose hydrolysis, preferential glucose utilization and the conversion of fructose to the undesirable by-products levan and sorbitol.

  20. Free-zone electrophoresis of animal cells. 1: Experiments on cell-cell interactions

    Science.gov (United States)

    Todd, P. W.; Hjerten, S.

    1985-01-01

    The electrophoretically migrating zones wasa monitored. The absence of fluid flows in the direction of migration permits direct measurement of electrophoretic velocities of any material. Sedimentation is orthogonal to electrokinetic motion and the effects of particle-particle interaction on electrophoretic mobility is studied by free zone electrophoresis. Fixed erythrocytes at high concentrations, mixtures of fixed erythrocytes from different animal species, and mixtures of cultured human cells were studied in low ionic strength buffers. The electrophoretic velocity of fixed erythrocytes was not altered by increasing cell concentration or by the mixing of erythrocytes from different species. When zones containing cultured human glial cells and neuroblastoma cells are permitted to interact during electrophoresis, altered migration patterns occur. It is found that cell-cell interactions depends upon cell type.

  1. ENHANCED DEGRADATION OF CAPTAN BY IMMOBILIZED CELLS OF BACILLUS CIRCULANS

    OpenAIRE

    Veena More; Preeti Tallur; More, Sunil S.; Niyonzima, Francois N.; Harichandra Ninnekar

    2014-01-01

    The possibility of using Bacillus circulans in degrading captan was evaluated by comparing the captan degradation rate by freely suspended and immobilized cells on agar, sodium alginate (SA), polyacrylamide (PA) and polyurethane-foam (PUF) in batch and repeated batch degradations. Under batch degradations, 50, 60, 72, and 88% of 0.1% captan was degraded by freely suspended cells, agar-, SA-, and PA-immobilized cells, respectively in 72 h; whereas 15, 47.5, 67.7 and 75% of 0.2% captan was degr...

  2. Covalent immobilization of p-selectin enhances cell rolling.

    Science.gov (United States)

    Hong, Seungpyo; Lee, Dooyoung; Zhang, Huanan; Zhang, Jennifer Q; Resvick, Jennifer N; Khademhosseini, Ali; King, Michael R; Langer, Robert; Karp, Jeffrey M

    2007-11-20

    Cell rolling is an important physiological and pathological process that is used to recruit specific cells in the bloodstream to a target tissue. This process may be exploited for biomedical applications to capture and separate specific cell types. One of the most commonly studied proteins that regulate cell rolling is P-selectin. By coating surfaces with this protein, biofunctional surfaces that induce cell rolling can be prepared. Although most immobilization methods have relied on physisorption, chemical immobilization has obvious advantages, including longer functional stability and better control over ligand density and orientation. Here we describe chemical methods to immobilize P-selectin covalently on glass substrates. The chemistry was categorized on the basis of the functional groups on modified glass substrates: amine, aldehyde, and epoxy. The prepared surfaces were first tested in a flow chamber by flowing microspheres functionalized with a cell surface carbohydrate (sialyl Lewis(x)) that binds to P-selectin. Adhesion bonds between P-selectin and sialyl Lewis(x) dissociate readily under shear forces, leading to cell rolling. P-selectin immobilized on the epoxy glass surfaces exhibited enhanced long-term stability of the function and better homogeneity as compared to that for surfaces prepared by other methods and physisorbed controls. The microsphere rolling results were confirmed in vitro with isolated human neutrophils. This work is essential for the future development of devices for isolating specific cell types based on cell rolling, which may be useful for hematologic cancers and certain metastatic cancer cells that are responsive to immobilized selectins.

  3. Decolorization of reactive Brilliant Blue KN-R by immobilized cells of Aspergillus ficuum

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Aspergillus ficuum was immobilized with sodium alginate, and decolorization of Reactive Brilliant Blue KN-R was studied on immobilized and free Aspergillus ficuum. The optimal preparation condition of the strain immobilization was obtained by the orthogonal test, it is sodium alginate 3%, CaCl2 5%, wet mycelia 30 g/L, calcific time 8 h. It was found that the immobilized cells could effectively decolorize Reactive Brilliant Blue KN-R, the optimum temperature and pH were 33℃ and 5.0, respectively. The kinetics study of decolorization of immobilized cells showed that the decolorization of Aspergillus ficuum immobilized conformed to zero-order reaction model. The decolorization efficiency of immobilized cell compared with that of free cell in different physical conditions. Results showed that the decolorization of immobilized cells with mycelia had the best efficiency. The immobilized cells could be reused after the first decolorization.

  4. Immobilized cell technology in beer brewing: Current experience and results

    Directory of Open Access Journals (Sweden)

    Leskošek-Čukalov Ida J.

    2005-01-01

    Full Text Available Immobilized cell technology (ICT has been attracting continual attention in the brewing industry over the past 30 years. Some of the reasons are: faster fermentation rates and increased volumetric productivity, compared to those of traditional beer production based on freely suspended cells, as well as the possibility of continuous operation. Nowadays, ICT technology is well established in secondary fermentation and alcohol- free and low-alcohol beer production. In main fermentation, the situation is more complex and this process is still under scrutiny on both the lab and pilot levels. The paper outlines the most important ICT processes developed for beer brewing and provides an overview of carrier materials, bioreactor design and examples of their industrial applications, as well as some recent results obtained by our research group. We investigated the possible applications of polyvinyl alcohol in the form of LentiKats®, as a potential porous matrices carrier for beer fermentation. Given are the results of growth studies of immobilized brewer's yeast Saccharomyces uvarum and the kinetic parameters obtained by using alginate microbeads with immobilized yeast cells and suspension of yeast cells as controls. The results indicate that the immobilization procedure in LentiKat® carriers has a negligible effect on cell viability and growth. The apparent specific growth rate of cells released in medium was comparable to that of freely suspended cells, implying preserved cell vitality. A series of batch fermentations performed in shaken flasks and an air-lift bioreactor indicated that the immobilized cells retained high fermentation activity. The full attenuation in green beer was reached after 48 hours in shaken flasks and less than 24 hours of fermentation in gas-lift bioreactors.

  5. Proteomic analysis of copper-binding proteins in excess copper-stressed rice roots by immobilized metal affinity chromatography and two-dimensional electrophoresis.

    Science.gov (United States)

    Song, Yufeng; Zhang, Hongxiao; Chen, Chen; Wang, Guiping; Zhuang, Kai; Cui, Jin; Shen, Zhenguo

    2014-04-01

    Copper (Cu) is an essential micronutrient required for plant growth and development. However, excess Cu can inactivate and disturb protein structure as a result of unavoidable binding to proteins. To understand better the mechanisms involved in Cu toxicity and tolerance in plants, we developed a new immobilized metal affinity chromatography (IMAC) method for the separation and isolation of Cu-binding proteins extracted from roots of rice seedling exposed to excess Cu. In our method, IDA-Sepharose or EDDS-Sepharose column (referred as pre-chromatography) and Cu-IDA-Sepharose column (referred as Cu-IMAC) were connected in tandem. Namely, protein samples were pre-chromatographed with IDA-Sepharose column to removal metal ions, then protein solution was flowed into Cu-IMAC column for enriching Cu-binding proteins in vitro. Compared with the control (Cu-IMAC without any pre-chromatography), IDA-Sepharose pre-chromatography method markedly increased yield of the Cu-IMAC-binding proteins, and number of protein spots and the abundance of 40 protein spots on two-dimensional electrophoresis (2-DE) gels. Thirteen protein spots randomly selected from 2-DE gel and 11 proteins were identified using MALDI-TOF-TOF MS. These putative Cu-binding proteins included those involved in antioxidant defense, carbohydrate metabolism, nucleic acid metabolism, protein folding and stabilization, protein transport and cell wall synthesis. Ten proteins contained one or more of nine putative metal-binding motifs reported by Smith et al. (J Proteome Res 3:834-840, 2004) and seven proteins contained one or two of top six motifs reported by Kung et al. (Proteomics 6:2746-2758, 2006). Results demonstrated that more proteins specifically bound with Cu-IMAC could be enriched through removal of metal ions from samples by IDA-Sepharose pre-chromatography. Further studies are needed on metal-binding characteristics of these proteins in vivo and the relationship between Cu ions and protein biological

  6. Plant cells: immobilization and oxygen transfer.

    NARCIS (Netherlands)

    Hulst, A.C.

    1987-01-01

    The study described in this thesis is part of the integrated project 'Biotechnological production of non-persistent bioinsecticides by means of plant cells invitro ' and was done in close cooperation with the research Institute Ital within the framework of NOVAPLANT. The plant cells us

  7. Bioreduction of chromate by immobilized cells of Halomonas sp

    Energy Technology Data Exchange (ETDEWEB)

    Murugavelh, S.; Mohanty, Kaustubha [Department of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati – 781039, Assam (India)

    2013-07-01

    In this work, the bioreduction of Cr(VI) by immobilized cells of Halomonas sp was reported. Ca alginate, acryl amide and agar were tested as the matrices for immobilization. Ca alginate was found to be the suitable matrix among the different matrices studied. Of the various dosages of inoculum studied 2 g/L was found to be the optimum. Glucose at 1 g L-1 was completely utilized by the immobilized Halomonas sp even in the presence of Cr(VI) at 40 mg L-1. The optimum pH for the bioreduction of Cr(VI) by immobilized Halomonas sp was found to be pH 6. The mechanical strength of the beads plays an essential role in the bioreduction process. Halomonas sp entrapped in a alginate matrix reported a maximum of 98.9 % of reduction for an initial Cr(VI) concentration of 10 mg L-1. The alginate beads can be reused for 3 times with slight drop in the percentage reduction. The presence of other metals decreased the bioreduction percentage.

  8. Production of high hydroxytyrosol yields via tyrosol conversion by Pseudomonas aeruginosa immobilized resting cells.

    Science.gov (United States)

    Bouallagui, Zouhaier; Sayadi, Sami

    2006-12-27

    An immobilized whole cell system was successfully performed to produce the most powerful antioxidant, hydroxytyrosol. Bioconversion of tyrosol into hydroxytyrosol was achieved via the immobilization of Pseudomonas aeruginosa resting cells in calcium alginate beads. Immobilization was advantageous as it allows immobilized cells to tolerate a greater tyrosol concentration than free cells. The bioconversion yield reached 86% in the presence of 5 g L-1 of tyrosol when cells immobilized in alginate beads were carried out in single batches. Evaluation of kinetic parameters showed the maintenance of the same catalytic efficiency expressed as Kcat/Km for both free and immobilized cells. The use of immobilized cells in repeated batches demonstrated a notable activity stabilization since the biocatalyst reusability was extended for at least four batches with a molar yield greater than 85%.

  9. [Study on CTP production from CMP by beer yeast cell immobilized in PVA].

    Science.gov (United States)

    Yang, Hong-Yi; Qian, Shi-Jun; Li, Gao-Wo

    2007-03-01

    With PVA as the carrier, the frozen beer yeast cells were immobilized for production of CTP from CMP. we explored the optimal condition of the immobilization from the aspects of the type, concentration of the PVA, and the immobilizing methods of cells In all 8 continuous batch of fermentation under the reactional condition of the immobilized cells, the conversion rate of CTP were maintained about 85% - 95%. Moreever, the storage stability of immobilized cells were investigated, and the products was also isolated and identifided by HPLC.

  10. Immobilization of microbial cell and yeast cell and its application to biomass conversion using radiation techniques

    Science.gov (United States)

    Kaetsu, Isao; Kumakura, Minoru; Fujimura, Takashi; Kasai, Noboru; Tamada, Masao

    The recent results of immobilization of cellulase-producing cells and ethanol-fermentation yeast by radiation were reported. The enzyme of cellulase produced by immobilized cells was used for saccharification of lignocellulosic wastes and immobilized yeast cells were used for fermentation reaction from glucose to ethanol. The wastes such as chaff and bagasse were treated by γ-ray or electron-beam irradiation in the presence of alkali and subsequent mechanical crushing, to form a fine powder less than 50 μm in diameter. On the other hand, Trichoderma reesei as a cellulase-producing microbial cell was immobilized on a fibrous carrier having a specific porous structure and cultured to produce cellulase. The enzymatic saccharification of the pretreated waste was carried out using the produced cellulase. The enhanced fermentation process to produce ethanol from glucose with the immobilized yeast by radiation was also studied. The ethanol productivity of immobilized growing yeast cells thus obtained was thirteen times that of free yeast cells in a 1:1 volume of liquid medium to immobilized yeast cells.

  11. Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae.

    Science.gov (United States)

    Najafpour, Ghasem; Younesi, Habibollah; Syahidah Ku Ismail, Ku

    2004-05-01

    Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process. The fermentation set-up was comprised of a column packed with beads of immobilized cells. The immobilization of S. cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase. The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate. The production of ethanol was steady after 24 h of operation. The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h. In addition, batch fermentation was carried out with 50 g/l glucose concentration. Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared. In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time. Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time. A yield of 38% was obtained with 150 g/l glucose. The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h. The cell growth rate was based on the Monod rate equation. The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively. The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively. Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively. The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh. Maximum production of ethanol in ICR when compared to batch reactor has shown to increase

  12. Immobilization of chlorine dioxide modified cells for uranium absorption.

    Science.gov (United States)

    He, Shengbin; Ruan, Binbiao; Zheng, Yueping; Zhou, Xiaobin; Xu, Xiaoping

    2014-11-01

    There has been a trend towards the use of microorganisms to recover metals from industrial wastewater, for which various methods have been reported to be used to improve microorganism adsorption characteristics such as absorption capacity, tolerance and reusability. In present study, chlorine dioxide(ClO2), a high-efficiency, low toxicity and environment-benign disinfectant, was first reported to be used for microorganism surface modification. The chlorine dioxide modified cells demonstrated a 10.1% higher uranium adsorption capacity than control ones. FTIR analysis indicated that several cell surface groups are involved in the uranium adsorption and cell surface modification. The modified cells were further immobilized on a carboxymethylcellulose(CMC) matrix to improve their reusability. The cell-immobilized adsorbent could be employed either in a high concentration system to move vast UO2(2+) ions or in a low concentration system to purify UO2(2+) contaminated water thoroughly, and could be repeatedly used in multiple adsorption-desorption cycles with about 90% adsorption capacity maintained after seven cycles.

  13. The application of single cell gel electrophoresis or comet assay to human monitoring studies

    Directory of Open Access Journals (Sweden)

    Valverde Mahara

    1999-01-01

    Full Text Available Objective. In the search of new human genotoxic biomarkers, the single cell gel electrophoresis assay has been proposed as a sensible alternative. Material and methods. This technique detects principally single strand breaks as well as alkali-labile and repair-retarded sites. Results. Herein we present our experience using the single cell gel electrophoresis assay in human population studies, both occupationally and environmentally exposed. Conclusions. We discuss the assay feasibility as a genotoxic biomarker.

  14. The application of single cell gel electrophoresis or comet assay to human monitoring studies

    OpenAIRE

    1999-01-01

    Objective. In the search of new human genotoxic biomarkers, the single cell gel electrophoresis assay has been proposed as a sensible alternative. Material and methods. This technique detects principally single strand breaks as well as alkali-labile and repair-retarded sites. Results. Herein we present our experience using the single cell gel electrophoresis assay in human population studies, both occupationally and environmentally exposed. Conclusions. We discuss the assay feasibility as a g...

  15. The production of sorbitol by permeabilized and immobilized cells of Z. mobilis in sucrose

    OpenAIRE

    Josiane Alessandra Vignoli; Maria Antonia Colabone Celligoi; Rui Sérgio Ferreira da Silva; Márcio de Barros

    2006-01-01

    The production of sorbitol by permeabilized and immobilized cells of Zymomonas mobilis in Luffa cylindrica was investigated in sucrose medium. A full 2³ factorial design was used to verify the influence of each factor and its interactions. The cell permeabilization showed a significant and negative effect upon the production of sorbitol, while the time of cultivation and the immobilization process were significant and positive. The results demonstrated that the cell immobilization and the tim...

  16. Transient Behavior of Ethanol Fermentation in Immobilized Cell Bioreactors*

    OpenAIRE

    Tohru, KANNO; Yoshinori, FUJISHIGE; Hiroyuki, Ito; koichi, yamazaki; Masayoshi, KOBAYASHI

    1990-01-01

    The dynamic behavior of ethanol fermentation catalysed by an immobilized cell has been studied in batch and continuous stirred tank bioreactors, changing the operating conditions in a stepwise fashion. The rate of ethanol fermentation in the flow reactor reaches a new steady state within 60 min for the stepwise change in temperature or flow rate at 15〜30℃ and the residence time t_R=40 hr. The rate of fermentation obeys the Lineweaven-Burk plot and the Michaelis constant is calculated

  17. Measurement of DNA damage in individual cells using the Single Cell Gel Electrophoresis (Comet) assay.

    Science.gov (United States)

    Hartley, Janet M; Spanswick, Victoria J; Hartley, John A

    2011-01-01

    The Single Cell Gel Electrophoresis (Comet) assay is a simple, versatile and sensitive method for measuring DNA damage in individual cells, allowing the determination of heterogeneity of response within a cell population. The basic alkaline technique described is for the determination of DNA strand break damage and its repair at a single cell level. Specific modifications to the method use a lower pH ('neutral' assay), or allow the measurement of DNA interstrand cross-links. It can be further adapted to, for example, study specific DNA repair mechanisms, be combined with fluorescent in situ hybridisation, or incorporate lesion specific enzymes.

  18. Growth and by-product profiles of Kluyveromyces marxianus cells immobilized in foamed alginate.

    Science.gov (United States)

    Wilkowska, Agnieszka; Kregiel, Dorota; Guneser, Onur; Karagul Yuceer, Yonca

    2015-01-01

    The aim of this research was to study how the yeast cell immobilization technique influences the growth and fermentation profiles of Kluyveromyces marxianus cultivated on apple/chokeberry and apple/cranberry pomaces. Encapsulation of the cells was performed by droplet formation from a foamed alginate solution. The growth and metabolic profiles were evaluated for both free and immobilized cells. Culture media with fruit waste produced good growth of free as well as immobilized yeast cells. The fermentation profiles of K. marxianus were different with each waste material. The most varied aroma profiles were noted for immobilized yeast cultivated on apple/chokeberry pomace.

  19. Two-dimensional gel electrophoresis analysis of the proteomes expressed in the human hepatoma cell line BEL-7404 and normal liver cell line L-02

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Proteome analysis technology has been used extensively in conducting discovery research of biology and has become one of the most essential technologies in functional genomics. The proteomes of the human hepatoma cell line BEL-7404 and the normal human liver cell line L-02 have been separated by high resolution two-dimensional gel electrophoresis (2-DE) with immobilized pH gradient isoelectric focusing (IPG-IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension (IPG-DALT). The resulting images have been analyzed using 2-D analysis software. Quantitative analysis reveals that 7 protein spots are detected only in hepatoma BEL-7404 cells, 14 only in L-02 cells, and 78 protein spots show significant fluctuation in quantity in both cell lines (P<0.01).These protein spots have been displayed on a proteome differential expression map. Analysis for the reproducibility of 2-DE indicates that the positional variability in the IEF dimension is 0.73 mm, while the variability in the SDS-PAGE dimension is 0.44 mm, and the quantitative variability is 17.6%-19.2%. These results suggest that the reproducibility of 2-DE has been suitable for the study of differential expression of proteomes. Proteome differential expression maps can be useful tools for disease diagnosis, drug-target validation analysis and biological process elucidation.

  20. Metal organic frameworks for enzyme immobilization in biofuel cells

    Science.gov (United States)

    Bodell, JaDee

    Interest in biofuel cells has been rapidly expanding as an ever-growing segment of the population gains access to electronic devices. The largest areas of growth for new populations using electronic devices are often in communities without electrical infrastructure. This lack of infrastructure in remote environments is one of the key driving factors behind the development of biofuel cells. Biofuel cells employ biological catalysts such as enzymes to catalyze oxidation and reduction reactions of select fuels to generate power. There are several benefits to using enzymes to catalyze reactions as compared to traditional fuel cells which use metal catalysts. First, enzymes are able to catalyze reactions at or near room temperature, whereas traditional metal catalysts are only efficient at very high temperatures. Second, biofuel cells can operate under mild pH conditions which is important for the eventual design of safe, commercially viable devices. Also, biofuel cells allow for implantable and flexible technologies. Finally, enzymes exhibit high selectivity and can be combined to fully oxidize or reduce the fuel which can generate several electrons from a single molecule of fuel, increasing the overall device efficiency. One of the main challenges which persist in biofuel cells is the instability of enzymes over time which tend to denature after hours or days. For a viable commercial biofuel cell to be produced, the stability of enzymes must be extended to months or years. Enzymes have been shown to have improved stability after being immobilized. The focus of this research was to find a metal organic framework (MOF) structure which could successfully immobilize enzymes while still allowing for electron transport to occur between the catalytic center of the enzyme and the electrode surface within a biofuel cell for power generation. Four MOF structures were successfully synthesized and were subsequently tested to determine the MOF's ability to immobilize the following

  1. Research on drug-receptor interactions and prediction of drug activity via oriented immobilized receptor capillary electrophoresis.

    Science.gov (United States)

    Liu, Chunye; Zhang, Xuejiao; Jing, Hui; Miao, Yanqing; Zhao, Lingzhi; Han, Yan; Cui, Cuixia

    2015-10-01

    Oriented covalent immobilized β2 -adrenergic receptor (β2 -AR) CE (OIRCE) was developed to determine the interactions between a set of natural extracts of Radix Paeoniae Rubra (NERPR) and β2 -AR, and to predict the activity of NERPR. The inner capillary surface is chemically bonded with stable β2 -AR coating via microwave-assisted technical synthesis. The modified capillaries were characterized via infrared spectroscopy and fluorescence microscopy. Furthermore, the bonding amounts of β2 -AR were first obtained via fluorescence spectroscopy method. In determining the amount of bonded β2 -AR, the regression equation A  =  576 707C + 35.449 and the correlation coefficient 0.9995 were obtained. This result revealed an excellent linear relationship in the range of 2 × 10(-4)  mg/mL to 1 × 10(-3)  mg/mL. The normalized capacity factor (KRCE ) was obtained using OIRCE in evaluating drug-receptor interactions. Related theories and equations were used to calculate KRCE values from apparent migration times of a solute and EOF. The order of KRCE and the binding constant (Kb ) values between drugs and β2 -AR was well consistent. The results confirmed that the OIRCE and KRCE values can be effectually used to investigate drug-receptor interactions, and OIRCE has the potential to predict drug activity and to select leading compounds from natural chemicals.

  2. Ethanol production from concentrated food waste hydrolysates with yeast cells immobilized on corn stalk.

    Science.gov (United States)

    Yan, Shoubao; Chen, Xiangsong; Wu, Jingyong; Wang, Pingchao

    2012-05-01

    The aim of the present study was to examine ethanol production from concentrated food waste hydrolysates using whole cells of S. cerevisiae immobilized on corn stalks. In order to improve cell immobilization efficiency, biological modification of the carrier was carried out by cellulase hydrolysis. The results show that proper modification of the carrier with cellulase hydrolysis was suitable for cell immobilization. The mechanism proposed, cellulase hydrolysis, not only increased the immobilized cell concentration, but also disrupted the sleek surface to become rough and porous, which enhanced ethanol production. In batch fermentation with an initial reducing sugar concentration of 202.64 ± 1.86 g/l, an optimal ethanol concentration of 87.91 ± 1.98 g/l was obtained using a modified corn stalk-immobilized cell system. The ethanol concentration produced by the immobilized cells was 6.9% higher than that produced by the free cells. Ethanol production in the 14th cycle repeated batch fermentation demonstrated the enhanced stability of the immobilized yeast cells. Under continuous fermentation in an immobilized cell reactor, the maximum ethanol concentration of 84.85 g/l, and the highest ethanol yield of 0.43 g/g (of reducing sugar) were achieved at hydraulic retention time (HRT) of 3.10 h, whereas the maximum volumetric ethanol productivity of 43.54 g/l/h was observed at a HRT of 1.55 h.

  3. MC3T3-E1 Cells on Titanium Surfaces with Nanometer Smoothness and Fibronectin Immobilization

    Directory of Open Access Journals (Sweden)

    Tohru Hayakawa

    2012-01-01

    Full Text Available The present study was aimed to evaluate the viability and total protein contents of osteoblast-like cells on the titanium surface with different surface mechanical treatment, namely, nanometer smoothing (Ra: approximately 2.0 nm and sandblasting (Ra: approximately 1.0 μm, and biochemical treatment, namely, with or without fibronectin immobilization. Fibronectin could be easily immobilized by tresyl chloride-activation technique. MC3T3-E1 cells were seeded on the different titanium surfaces. Cell viability was determined by MTT assay. At 1 day of cell culture, there were no significant differences in cell viability among four different titanium surfaces. At 11 days, sandblasted titanium surface with fibronectin immobilization showed the significantly highest cell viability than other titanium surface. No significant differences existed for total protein contents among four different titanium surfaces at 11 days of cell culture. Scanning electron microscopy observation revealed that smoothness of titanium surface produced more spread cell morphologies, but that fibronectin immobilization did not cause any changes of the morphologies of attached cells. Fibronectin immobilization provided greater amount of the number of attached cells and better arrangement of attached cells. In conclusion, the combination of sandblasting and fibronectin immobilization enhanced the cell viability and fibronectin immobilization providing better arrangements of attached cells.

  4. Assay of Histamine in Single Mast Cells by Capillary Zone Electrophoresis with Electrochemical Detection

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Capillary zone electrophoresis was employed for the analysis of histamine in single rat peritoneal mast cells using an amperometric detector. In this method, individual mast cells and then 0.02 mol/L NaOH as a lysing solution are injected into the front end of the separation capillary. A cell injector was constructed for easy injection of single cells. Histamine in single mast cells has been identified and quantified.

  5. Long-term repeated biodesulfurization by immobilized Rhodococcus erythropolis KA2-5-1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Naito, M.; Kawamoto, T.; Tanaka, A. [Kyoto Univ., Yoshida (Japan). Dept. of Synthetic Chemistry and Biological Chemistry; Fujino, K.; Kobayashi, M.; Maruhashi, K. [Advanced Technology and Research Inst., Petroleum Energy Center, Shizuoka (Japan)

    2001-07-01

    In this study, biodesulfurization (BDS) was carried out using immobilized Rhodococcus erythropolis KA2-5-1 in n-tetradecane containing dibenzothiophene (DBT) as a model oil (n-tetradecane/immobilized cell biphasic system). The cells were immobilized by entrapping them with calcium alginate, agar, photo-crosslinkable resin prepolymers (ENT-4000 and ENTP-4000), and urethane prepolymers (PU-3 and PU-6); and it was found that ENT-4000-immobilized cells had the highest DBT desulfurization activity in the model oil system without leakage of cells from the support. Furthermore, ENT-4000-immobilized cells could catalyze BDS repeatedly in this system for more than 900 h with reactivation; and recovery of both the biocatalyst and the desulfurized model oil was easy. This study would give a solution to the problems in BDS, such as the troublesome process of recovering desulfurized oil and the short life of BDS biocatalysts. (orig.)

  6. Biodegradation of phenol by using free and immobilized cells of Acinetobacter sp. BS8Y.

    Science.gov (United States)

    Jiang, Lichun; Ruan, Qiping; Li, Rulan; Li, Tiandong

    2013-03-01

    Strain BS8Y with high biodegradation activity and high tolerance of phenol was isolated from activated sludge in an insulating material plant of China. This strain was capable of removing 99.2% of the initial 600 mg/l phenol in liquid minimal medium within 24 h and tolerating phenol at concentrations of up to 1,200 mg/ml. DNA sequencing and homologous analysis of the 16S rRNA gene identified that the strain BS8Y belonged to an Acinetobacter species. Polyvinyl alcohol was used as gel matrix to immobilize the strain BS8Y. The factors affecting the phenol degradation by immobilized cells and the phenol removal efficiency of free and immobilized cells were investigated; the stability of the immobilized cells is also reported. The results show that the immobilized cells could tolerate a higher phenol level and protected the bacteria much more effectively against changes in temperature and pH. The phenol degradation efficiency was high at up to 96% within 30 h, with an initial concentration of 800 mg/l phenol, and the immobilized cells showed better performance than the suspended cells. Reusability tests revealed that the immobilized cells were stable enough even after reuse for ten times or storing at 4°C for 35 d. These results demonstrate that immobilized Acinetobacter sp. BS8Y possesses a good application potential in the treatment of phenol-containing wastewater.

  7. Formaldehyde degradation by Ralstonia eutropha in an immobilized cell bioreactor.

    Science.gov (United States)

    Habibi, Alireza; Vahabzadeh, Farzaneh

    2013-01-01

    The formaldehyde (FA) degradation ability of the loofa-immobilized Ralstonia eutropha cells in a packed bed reactor was modeled using a statistically based design of the experiment (DOE) considering application of response surface methodology (RSM). The simultaneous effects of four operative test factors on the cells performance in terms of FA degradation rate and extent of the chemical oxygen demand (COD) removal were monitored. The combination of factors at initial FA concentration of 629.7 mg L(-1)h(-1), recycling substrate flow rate of 4.4 mL min(-1), aeration rate of 1.05 vvm, and the system's temperature of 28.8°C resulted the optimal conditions for the FA biodegradation rate and COD removal efficiency. Loofa porous structure was found to be a protective environment for the cells in exposing to the toxic substances and the scanning electron microscopy (SEM) images revealed extensive cells penetration within this support. Oxygen transfer analysis in the form of evaluating K la value was also carried out and at the optimum conditions of the DOE was equaled to 9.96 h(-1)and oxygen uptake rate was 35.6 mg L(-1)h(-1).

  8. Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1

    Directory of Open Access Journals (Sweden)

    Preeti N. Tallur

    2015-09-01

    Full Text Available Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF, polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water.

  9. Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1.

    Science.gov (United States)

    Tallur, Preeti N; Mulla, Sikandar I; Megadi, Veena B; Talwar, Manjunatha P; Ninnekar, Harichandra Z

    2015-01-01

    Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF), polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM) than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water.

  10. DNA sequencing with capillary electrophoresis and single cell analysis with mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Fung, N.

    1998-03-27

    Since the first demonstration of the laser in the 1960`s, lasers have found numerous applications in analytical chemistry. In this work, two different applications are described, namely, DNA sequencing with capillary gel electrophoresis and single cell analysis with mass spectrometry. Two projects are described in which high-speed DNA separations with capillary gel electrophoresis were demonstrated. In the third project, flow cytometry and mass spectrometry were coupled via a laser vaporization/ionization interface and individual mammalian cells were analyzed. First, DNA Sanger fragments were separated by capillary gel electrophoresis. A separation speed of 20 basepairs per minute was demonstrated with a mixed poly(ethylene oxide) (PEO) sieving solution. In addition, a new capillary wall treatment protocol was developed in which bare (or uncoated) capillaries can be used in DNA sequencing. Second, a temperature programming scheme was used to separate DNA Sanger fragments. Third, flow cytometry and mass spectrometry were coupled with a laser vaporization/ionization interface.

  11. Degradation of h-acid by free and immobilized cells of Alcaligenes latus

    Directory of Open Access Journals (Sweden)

    M.S. Usha

    2010-12-01

    Full Text Available Alcaligenes latus, isolated from industrial effluent, was able to grow in mineral salts medium with 50 ppm (0.15 mM of H-acid as a sole source of carbon. Immobilization of Alcaligenes latus in Ca-alginate and polyurethane foam resulted in cells embedded in the matrices. When free cells and immobilized cells were used for biodegradation studies at concentration ranging from 100 ppm (0.3 mM to 500 ppm (1.15 mM degradation rate was enhanced with immobilized cells. Cells immobilized in polyurethane foam showed 100% degradation up to 350 ppm (1.05 mM and 57% degradation at 500 ppm (1.5 mM. Degradation rate of Ca-alginate immobilized cells was less as compared to that of polyurethane foam immobilized cells. With Ca-alginate immobilized cells 100% degradation was recorded up to 200 ppm (0.6 mM of H-acid and only 33% degradation was recorded at 500 ppm (1.5 mM of H-acid. Spectral analysis of the products after H-acid utilization showed that the spent medium did not contain any aromatic compounds indicating H-acid degradation by A. latus.

  12. Comparison of Di-n-methyl Phthalate Biodegradation by Free and Immobilized Microbial Cells

    Institute of Scientific and Technical Information of China (English)

    JIAN-LONG WANG; YU-CAI YE; WEI-ZHONG WU

    2003-01-01

    Objective To compare the biodegradation of di-n-methyl pathalate by free and immobilizedmicrobial cells. Methods The enrichment and isolation technique was used to isolate themicroorganism. The PAV-entrapment method was utilized to immobilize the microorganisms. Thescanning electron microscophy (SEM) was used to observe the growth and distribution of microbialcells immobilized inside the PVA bead gels. The GC/MS method was used to identify the mainintermediates of DMP degradation. Results The microbial cells could grow quite well in PVA gel.The metabolic pathway did not change before and after immobilization of the microbial cells. Thedegradation rate of immobilized cells was higher than that of free cells. Conclusion Theimmobilized microbial cells possess advantages than free cells when applied to the biodegradation oftoxic organic pollutants.

  13. Development of High-Productivity Continuous Ethanol Production using PVA-Immobilized Zymomonas mobilis in an Immobilized-Cells Fermenter

    Directory of Open Access Journals (Sweden)

    Nurhayati Nurhayati

    2015-07-01

    Full Text Available Ethanol as one of renewable energy was being considered an excellent alternative clean-burning fuel to replace gasoline. Continuous ethanol fermentation systems had offered important economic advantages compared to traditional systems. Fermentation rates were significantly improved, especially when continuous fermentation was integrated with cell immobilization techniques to enrich the cells concentration in fermentor. Growing cells of Zymomonas mobilis immobilized in polyvinyl alcohol (PVA gel beads were employed in an immobilized-cells fermentor for continuous ethanol fermentation from glucose. The glucose loading, dilution rate, and cells loading were varied in order to determine which best condition employed in obtaining both high ethanol production and low residual glucose with high dilution rate. In this study, 20 g/L, 100 g/L, 125 g/L and 150 g/L of glucose concentration and 20% (w/v, 40% (w/v and 50% (w/v of cells loading were employed with range of dilution rate at 0.25 to 1 h-1. The most stable production was obtained for 25 days by employing 100 g/L of glucose loading. Meanwhile, the results also exhibited that 125 g/L of glucose loading as well as 40% (w/v of cells loading yielded high ethanol concentration, high ethanol productivity, and acceptable residual glucose at 62.97 g/L, 15.74 g/L/h and 0.16 g/L, respectively. Furthermore, the dilution rate of 4 hour with 100 g/L and 40% (w/v of glucose and cells loading was considered as the optimum condition with ethanol production, ethanol productivity and residual glucose obtained were 49.89 g/L, 12.47 g/L/h, and 2.04 g/L, respectively. This recent study investigated ethanol inhibition as well. The present research had proved that high sugar concentration was successfully converted to ethanol. These achieved results were promising for further study.

  14. Phospholipid polymer-based antibody immobilization for cell rolling surfaces in stem cell purification system.

    Science.gov (United States)

    Mahara, Atsushi; Chen, Hao; Ishihara, Kazuhiko; Yamaoka, Tetsuji

    2014-01-01

    We previously developed an antibody-conjugated cell rolling column that successfully separates stem cell subpopulations depending on the cell surface marker density, but a large amount of the injected cells were retained in the column because of non-specific interactions. In this study, an amphiphilic copolymer, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (nBMA)-co-N-vinyl formamide (NVf)], with phospholipid polar side groups was designed as a novel antibody-immobilizing modifier. The formamide groups in NVf units were converted to active maleimide groups. A plastic flow microfluidic chamber was coated with the copolymers, and a reduced anti-CD90 antibody was immobilized. The adipose tissue-derived stem cells isolated from the rat were injected into the flow chamber, and their rolling behavior was observed under a microscope with a high-speed camera. Non-specific cell adhesion was reduced strongly by means of this immobilization method because of the MPC unit, resulting in a high percentage of rolling cells. These results demonstrate that a surface coated with phospholipid polar groups can be used in an effective stem cell separation system based on the cell rolling process.

  15. Application of Klebsiella oxytoca immobilized cells on the treatment of cyanide wastewater.

    Science.gov (United States)

    Chen, C Y; Kao, C M; Chen, S C

    2008-03-01

    Klebsiella oxytoca, isolated from cyanide-containing industrial wastewater, has been shown to be able to biodegrade cyanide to non-toxic end products. The technology of immobilized cells can be applied in biological treatment to enhance the efficiency and effectiveness of biodegradation. In this study, potassium cyanide was used as the target compound and both alginate and cellulose triacetate techniques were applied for the preparation of immobilized cells. Results from this study show that KCN can be utilized as the sole nitrogen source by K. oxytoca. The free suspension systems reveal that the cell viability was highly affected by initial KCN concentration and pH. Results show that immobilized cell systems could tolerate a higher level of KCN concentration and wider ranges of pH. In the batch experiments, the maximum KCN removal efficiencies using alginate and cellulose triacetate immobilized beads were 0.108 and 0.101mM h(-1) at pH 7, respectively. Results also indicate that immobilized system can support a higher biomass concentration. Complete KCN degradation was observed after the operation of four consecutive degradation experiments with the same batch of immobilized cells. This suggests that the activity of immobilized cells can be maintained and KCN can be used as the nitrogen source throughout KCN degradation experiments. The maximum KCN removal rates using alginate and cellulose triacetate immobilized beads in continuous-column system were 0.224 and 0.192mMh(-1) with initial KCN concentration of 3mM, respectively. Results indicate that the immobilized cells of K. oxytoca would be applicable to the treatment of cyanide-containing wastewaters.

  16. Hydrophilic PCU scaffolds prepared by grafting PEGMA and immobilizing gelatin to enhance cell adhesion and proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Changcan; Yuan, Wenjie; Khan, Musammir; Li, Qian [School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Feng, Yakai, E-mail: yakaifeng@tju.edu.cn [School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin 300072 (China); Tianjin University-Helmholtz-Zentrum Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, Tianjin 300072 (China); Collaborative Innovation Center of Chemical Science and Chemical Engineering (Tianjin) Tianjin 300072 (China); Yao, Fanglian [School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin 300072 (China); Tianjin University-Helmholtz-Zentrum Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, Tianjin 300072 (China); Zhang, Wencheng, E-mail: wenchengzhang@yahoo.com [Department of Physiology and Pathophysiology, Logistics University of Chinese People' s Armed Police Force, Tianjin 300162 (China)

    2015-05-01

    Gelatin contains many functional motifs which can modulate cell specific adhesion, so we modified polycarbonate urethane (PCU) scaffold surface by immobilization of gelatin. PCU-g-gelatin scaffolds were prepared by direct immobilizing gelatins onto the surface of aminated PCU scaffolds. To increase the immobilization amount of gelatin, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto PCU scaffolds by surface initiated atom transfer radical polymerization. Then, following amination and immobilization, PCU-g-PEGMA-g-gelatin scaffolds were obtained. Both modified scaffolds were characterized by chemical and biological methods. After immobilization of gelatin, the microfiber surface became rough, but the original morphology of scaffolds was maintained successfully. PCU-g-PEGMA-g-gelatin scaffolds were more hydrophilic than PCU-g-gelatin scaffolds. Because hydrophilic PEGMA and gelatin were grafted and immobilized onto the surface, the PCU-g-PEGMA-g-gelatin scaffolds showed low platelet adhesion, perfect anti-hemolytic activity and excellent cell growth and proliferation capacity. It could be envisioned that PCU-g-PEGMA-g-gelatin scaffolds might have potential applications in tissue engineering artificial scaffolds. - Graphical abstract: PCU-g-gelatin scaffolds were prepared by direct immobilizing gelatin onto the surface of aminated PCU scaffolds (method a). To increase the immobilization amount of gelatin, PEGMAs were grafted onto the scaffold surface by SI-ATRP. PCU-g-PEGMA-g-gelatin scaffolds were prepared by method b. The gelatin modified scaffolds exhibited high hydrophilicity, low platelet adhesion, perfect anti-hemolytic activity, and excellent cell adhesion and proliferation capacity. They might have potential applications as tissue engineering scaffolds for artificial blood vessels. - Highlights: • Hydrophilic scaffolds were prepared by grafting PEGMA and immobilization of gelatins. • Grafting PEGMA enhanced the immobilization amount of gelatin

  17. Micromagnetic Cancer Cell Immobilization and Release for Real-Time Single Cell Analysis

    Science.gov (United States)

    Jaiswal, Devina; Rad, Armin Tahmasbi; Nieh, Mu-Ping; Claffey, Kevin P.; Hoshino, Kazunori

    2017-04-01

    Understanding the interaction of live cells with macromolecules is crucial for designing efficient therapies. Considering the functional heterogeneity found in cancer cells, real-time single cell analysis is necessary to characterize responses. In this study, we have designed and fabricated a microfluidic channel with patterned micromagnets which can temporarily immobilize the cells during analysis and release them after measurements. The microchannel is composed of plain coverslip top and bottom panels to facilitate easy microscopic observation and undisturbed application of analytes to the cells. Cells labeled with functionalized magnetic beads were immobilized in the device with an efficiency of 90.8±3.6%. Since the micromagnets are made of soft magnetic material (Ni), they released cells when external magnetic field was turned off from the channel. This allows the reuse of the channel for a new sample. As a model drug analysis, the immobilized breast cancer cells (MCF7) were exposed to fluorescent lipid nanoparticles and association and dissociation were measured through fluorescence analysis. Two concentrations of nanoparticles, 0.06 μg/ml and 0.08 μg/ml were tested and time lapse images were recorded and analyzed. The microfluidic device was able to provide a microenvironment for sample analysis, making it an efficient platform for real-time analysis.

  18. "Fish-in-net", a novel method for cell immobilization of Zymomonas mobilis.

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    Xuedun Niu

    Full Text Available BACKGROUND: Inorganic mesoporous materials exhibit good biocompatibility and hydrothermal stability for cell immobilization. However, it is difficult to encapsulate living cells under mild conditions, and new strategies for cell immobilization are needed. We designed a "fish-in-net" approach for encapsulation of enzymes in ordered mesoporous silica under mild conditions. The main objective of this study is to demonstrate the potential of this approach in immobilization of living cells. METHODOLOGY/PRINCIPAL FINDINGS: Zymomonas mobilis cells were encapsulated in mesoporous silica-based materials under mild conditions by using a "fish-in-net" approach. During the encapsulation process, polyethyleneglycol was used as an additive to improve the immobilization efficiency. After encapsulation, the pore size, morphology and other features were characterized by various methods, including scanning electron microscopy, nitrogen adsorption-desorption analysis, transmission electron microscopy, fourier transform infrared spectroscopy, and elemental analysis. Furthermore, the capacity of ethanol production by immobilized Zymomonas mobilis and free Zymomonas mobilis was compared. CONCLUSIONS/SIGNIFICANCE: In this study, Zymomonas mobilis cells were successfully encapsulated in mesoporous silica-based materials under mild conditions by the "fish-in-net" approach. Encapsulated cells could perform normal metabolism and exhibited excellent reusability. The results presented here illustrate the enormous potential of the "fish-in-net" approach for immobilization of living cells.

  19. Combinational Effect of Cell Adhesion Biomolecules and Their Immobilized Polymer Property to Enhance Cell-Selective Adhesion

    Directory of Open Access Journals (Sweden)

    Rio Kurimoto

    2016-01-01

    Full Text Available Although surface immobilization of medical devices with bioactive molecules is one of the most widely used strategies to improve biocompatibility, the physicochemical properties of the biomaterials significantly impact the activity of the immobilized molecules. Herein we investigate the combinational effects of cell-selective biomolecules and the hydrophobicity/hydrophilicity of the polymeric substrate on selective adhesion of endothelial cells (ECs, fibroblasts (FBs, and smooth muscle cells (SMCs. To control the polymeric substrate, biomolecules are immobilized on thermoresponsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide (poly(NIPAAm-co-CIPAAm-grafted glass surfaces. By switching the molecular conformation of the biomolecule-immobilized polymers, the cell-selective adhesion performances are evaluated. In case of RGDS (Arg-Gly-Asp-Ser peptide-immobilized surfaces, all cell types adhere well regardless of the surface hydrophobicity. On the other hand, a tri-Arg-immobilized surface exhibits FB-selectivity when the surface is hydrophilic. Additionally, a tri-Ile-immobilized surface exhibits EC-selective cell adhesion when the surface is hydrophobic. We believe that the proposed concept, which is used to investigate the biomolecule-immobilized surface combination, is important to produce new biomaterials, which are highly demanded for medical implants and tissue engineering.

  20. ENHANCED PRODUCTION OF PECTINOLYTIC ENZYMES FROM IMMOBILIZED CELLS OF MIXED ASPERGILLUS SPECIES

    Directory of Open Access Journals (Sweden)

    Shruti Singh

    2012-12-01

    Full Text Available The cells of isolated mixed culture of Aspergillus fumigatus and Aspergillus sydowii were immobilized in calcium alginate beads. Studies were carried out on different parameters like alginate concentration, incubation time and bead inoculum which affects the productivity and stability of the immobilized system. The best enzymatic activities were obtained with 3% alginate concentration, 48h of incubation time and 200 beads/flask of inoculum. Optimization of these factors causes an increase in enzymatic activities and the possibility of semicontinuous cultivation. Immobilized cells could be reused in five successive reaction cycles with a slight decrease in activities.

  1. Cell immobilization on polymer by air atmospheric pressure plasma jet treatment

    Science.gov (United States)

    Lee, Jung-Hwan; Kwon, Jae-Sung; Om, Ji-yeon; Kim, Yong-Hee; Choi, Eun-Ha; Kim, Kwang-Mahn; Kim, Kyoung-Nam

    2014-08-01

    The study of cell immobilization on delicate polymer by an air atmospheric pressure plasma jet (AAPPJ) is required for its medical application. The aim of this study was to evaluate whether AAPPJ treatment induce cell immobilization effect on delicate polymers without significant change of surface roughness by AAPPJ treatment. After surface roughness, dynamic contact angle, and chemical characteristics were investigated, the immobilization effect was evaluated with the mouse fibroblast L929 cell line. Surface roughness change was not observed (P > 0.05) in either delicate dental wax or polystyrene plate (PSP) as advancing and receding contact angles significantly decreased (P < 0.05), thanks to decreased hydrocarbon and formation of oxygen-related functional groups in treated PSP. Adherent L929 cells with elongated morphology were found in treated PSP along with the formation of immobilization markers vinculin and actin cytoskeleton. Increased PTK2 gene expression upregulated these markers on treated PSP.

  2. Comparative analysis of hydrogen-producing bacteria and its immobilized cells for characteristics of hydrogen production

    Institute of Scientific and Technical Information of China (English)

    王相晶; 任南琪; 向文胜; 王爱杰; 林明; 郭婉茜

    2003-01-01

    A strain of hydrogen producing bacteria was immobilized by polyvinyl alcohol-boric acid method,with the addition of a small amount of calcium alginate. The immobilized cells were insensitive to the presence of traces of O2. Moreover, the immobilized cells increased both the evolution rate and the yield of hydrogen production. Batch experiments with a medium containing 10 g/L glucose demonstrated the yields of hydrogen production by the immobilized and free cells were 2.14 mol/mol glucose and 1.69 mol/mol glucose, respectively.In continuous cultures atmedium retention time of 2. 0 h, the yield and the evolution rate of hydrogen producmedium retention time of 6. 0 h, the yield and the evolution rate of hydrogen production by free cells were only 1.75 mol/mol glucose and 362.9ml/(L·h),respectively.

  3. Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test.

    Science.gov (United States)

    Su, Yan; Gao, Lijun; Ma, Qiang; Zhou, Lishe; Qin, Liangyi; Han, Lihong; Qin, Wenbin

    2010-09-01

    To elucidate the protein-protein interactions of hemoglobin (Hb) variants A and A(2), HbA was first shown to bind with HbA(2) in live red blood cells (RBCs) by diagonal electrophoresis and then the interaction between HbA and HbA(2) outside the RBC was shown by cross electrophoresis. The starch-agarose gel electrophoresis of hemolysate, RBCs, freeze-thawed RBCs and the supernatant of freeze-thawed RBCs showed that the interaction between HbA and HbA(2) was affected by membrane integrity. To identify the proteins involved in the interaction, protein components located between HbA and HbA(2) in RBCs (RBC HbA-HbA(2)) and hemolysate (hemolysate HbA-HbA(2)) were isolated from the starch-agarose gel and separated by 5-12% SDS-PAGE. The results showed that there was a ≈22 kDa protein band located in the RBC HbA-HbA(2) but not in hemolysate HbA-HbA(2). Sequencing by LC/MS/MS showed that this band was a protein complex that included mainly thioredoxin peroxidase B, α-globin, δ-globin and β-globin. Thus, using our unique in vivo whole blood cell electrophoresis release test, Hbs were proven for the first time to interact with other proteins in the live RBC.

  4. Optimized immobilization of lectins using self-assembled monolayers on polysilicon encoded materials for cell tagging.

    Science.gov (United States)

    Penon, Oriol; Siapkas, Dimitrios; Novo, Sergi; Durán, Sara; Oncins, Gerard; Errachid, Abdelhamid; Barrios, Lleonard; Nogués, Carme; Duch, Marta; Plaza, José Antonio; Pérez-García, Lluïsa

    2014-04-01

    Self-assembled monolayers (SAMs) have been used for the preparation of functional microtools consisting of encoded polysilicon barcodes biofunctionalized with proteins of the lectin family. These hybrid microtools exploit the lectins ability for recognizing specific carbohydrates of the cell membrane to give an efficient system for cell tagging. This work describes how the control of the methodology for SAM formation on polysilicon surfaces followed by lectin immobilization has a crucial influence on the microtool biofunction. Several parameters (silanization time, silane molar concentration, type of solvent or deposition methodology) have been studied to establish optimal function. Furthermore, silanes incorporating different terminal groups, such as aldehyde, activated ester or epoxide groups were tested in order to analyze their chemical coupling with the biomolecules, as well as their influence on the biofunctionality of the immobilized protein. Two different lectins - wheat germ agglutinin (WGA) and phytohemagglutinin (PHA-L) - were immobilized, because they have different and specific cell recognition behaviour and exhibit different cell toxicity. In this way we can assess the effect of intrinsic bulk toxicity with that of the cell compatibility once immobilized as well as the importance of cell affinity. A variety of nanometrical techniques were used to characterize the active surfaces, and lectin immobilization was quantified using ultraviolet-visible absorption spectroscopy (UV-vis) and optical waveguide light mode spectroscopy (OWLS). Once the best protocol was found, WGA and PHA were immobilized on polysilicon coded barcodes, and these microtools showed excellent cell tagging on living mouse embryos when WGA was used.

  5. The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay.

    Science.gov (United States)

    Hellman, B; Vaghef, H; Boström, B

    1995-03-01

    Single cell gel electrophoresis under alkaline conditions is a technique used to detect primary DNA damage in individual mammalian cells. Cells embedded in agarose on microscope slides are subjected to lysis, unwinding of DNA and electrophoresis at high pH. After staining with a fluorescent dye, cells with DNA damage display increased migration of genetic material from the cell nucleus. The damage is quantified by measuring the displacement between the genetic material of the nucleus ('comet head') and the resulting 'tail'. The torsional moment of the tail ('tail moment') has been suggested to be an appropriate index of induced DNA damage in considering both the migration of the genetic material as well as the relative amount of DNA in the tail. In the present paper it will be shown that the moment of inertia ('tail inertia'), a not previously described tail parameter, provides a more precise description of the distribution of individual DNA fragments within the tails. The tail inertia was also found to be the most sensitive indicator of the DNA damage induced in peripheral lymphocytes from mice given a single intraperitoneal injection of cyclophosphamide (150 mg/kg b.w.). It is concluded that the tail inertia is an important complement to other tail parameters when looking for damage of DNA with the single cell gel electrophoresis assay.

  6. Recent insights into the cell immobilization technology applied for dark fermentative hydrogen production.

    Science.gov (United States)

    Kumar, Gopalakrishnan; Mudhoo, Ackmez; Sivagurunathan, Periyasamy; Nagarajan, Dillirani; Ghimire, Anish; Lay, Chyi-How; Lin, Chiu-Yue; Lee, Duu-Jong; Chang, Jo-Shu

    2016-11-01

    The contribution and insights of the immobilization technology in the recent years with regards to the generation of (bio)hydrogen via dark fermentation have been reviewed. The types of immobilization practices, such as entrapment, encapsulation and adsorption, are discussed. Materials and carriers used for cell immobilization are also comprehensively surveyed. New development of nano-based immobilization and nano-materials has been highlighted pertaining to the specific subject of this review. The microorganisms and the type of carbon sources applied in the dark hydrogen fermentation are also discussed and summarized. In addition, the essential components of process operation and reactor configuration using immobilized microbial cultures in the design of varieties of bioreactors (such as fixed bed reactor, CSTR and UASB) are spotlighted. Finally, suggestions and future directions of this field are provided to assist the development of efficient, economical and sustainable hydrogen production technologies.

  7. Evaluation of genotoxicity of the acute gamma radiation on earthworm Eisenia fetida using single cell gel electrophoresis technique (Comet assay).

    Science.gov (United States)

    Sowmithra, K; Shetty, N J; Jha, S K; Chaubey, R C

    2015-12-01

    Earthworms (Eisenia fetida) most suitable biological indicators of radioactive pollution. Radiation-induced lesions in DNA can be considered to be molecular markers for early effects of ionizing radiation. Gamma radiation produces a wide spectrum of DNA. Some of these lesions, i.e., DNA strand breaks and alkali labile sites can be detected by the single-cell gel electrophoresis (SCGE) or comet assay by measuring the migration of DNA from immobilized nuclear DNA. E. fetida were exposed to different doses of gamma radiation, i.e., 1, 5, 10, 20, 30, 40 and 50Gy, and comet assay was performed for all the doses along with control at 1, 3 and 5h post irradiation to evaluate the genotoxicity of gamma radiation in this organism. The DNA damage was measured as percentage of comet tail DNA. A significant increase in DNA damage was observed in samples exposed to 5Gy and above, and the increase in DNA damage was dose dependent i.e., DNA damage was increased with increased doses of radiation. The highest DNA damage was noticed at 1h post irradiation and gradually decreased with time, i.e., at 3 and 5h post irradiation. The present study reveals that gamma radiation induces DNA damage in E. fetida and the comet assay is a sensitive and rapid method for its detection to detect genotoxicity of gamma radiation.

  8. Engineering cholesterol-based fibers for antibody immobilization and cell capture

    Science.gov (United States)

    Cohn, Celine

    In 2015, the United States is expected to have nearly 600,000 deaths attributed to cancer. Of these 600,000 deaths, 90% will be a direct result of cancer metastasis, the spread of cancer throughout the body. During cancer metastasis, circulating tumor cells (CTCs) are shed from primary tumors and migrate through bodily fluids, establishing secondary cancer sites. As cancer metastasis is incredibly lethal, there is a growing emphasis on developing "liquid biopsies" that can screen peripheral blood, search for and identify CTCs. One popular method for capturing CTCs is the use of a detection platform with antibodies specifically suited to recognize and capture cancer cells. These antibodies are immobilized onto the platform and can then bind and capture cells of interest. However, current means to immobilize antibodies often leave them with drastically reduced function. The antibodies are left poorly suited for cell capture, resulting in low cell capture efficiencies. This body of work investigates the use of lipid-based fibers to immobilize proteins in a way that retains protein function, ultimately leading to increased cell capture efficiencies. The resulting increased efficiencies are thought to arise from the retained three-dimensional structure of the protein as well as having a complete coating of the material surface with antibodies that are capable of interacting with their antigens. It is possible to electrospin cholesterol-based fibers that are similar in design to the natural cell membrane, providing proteins a more natural setting during immobilization. Such fibers have been produced from cholesterol-based cholesteryl succinyl silane (CSS). These fibers have previously illustrated a keen aptitude for retaining protein function and increasing cell capture. Herein the work focuses on three key concepts. First, a model is developed to understand the immobilization mechanism used by electrospun CSS fibers. The antibody immobilization and cell capturing

  9. Protein Electrophoresis/Immunofixation Electrophoresis

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Protein Electrophoresis Immunofixation Electrophoresis Share this page: Was this page helpful? Also known as: Serum Protein Electrophoresis; Protein ELP; SPE; SPEP; Urine Protein Electrophoresis; ...

  10. [Study on immobilized cells for producing alpha-amylase by using polyving alcohol as the carrier(II): The effect of fermentating conditions on the ability producing alpha-amylase of the cells immobilized with polyving alcohol as the corrier and continuous fermentation of the immobilized cells in CSTR].

    Science.gov (United States)

    Liu, Z; Wang, J; Li, Z

    1998-03-01

    The effects of fermentating conditions on the ability of immobilized cells with PVA as carrier for producing alpha-amylase were studied. The continuous fermentation with the immobilized cells were tested in continuous flow stirred tank reactor (CSTR). The results showed that the adaptability of the immobilized Bacillus substilis to pH increased after immobilization. In CSTR, the immobilized cells can be fermentated continuously for 360 hrs and the activity of alpha-amylase can be kept on the level of about 170 u/ml.

  11. Continuous ethanol production from pineapple cannery waste using immobilized yeast cells.

    Science.gov (United States)

    Nigam, J N

    2000-06-23

    The cells of Saccharomyces cerevisiae ATCC 24553, were immobilized in k-carrageenan and packed in a tapered glass column reactor for ethanol production from pineapple cannery waste at temperature 30 degrees C and pH 4.5. The maximum productivity was 42.8 g ethanol 1(-1) h(-1) at a dilution rate of 1.5 h(-1). The volumetric ethanol productivity of the immobilized cells was ca. 11.5 times higher than the free cells. The immobilized cell reactor was operated over a period of 87 days at a dilution rate of 1.0 h(-1), without any loss in the immobilized cell activity. The maximum specific ethanol productivity and specific sugar uptake rate of the immobilized cells were 1.2 g ethanol g(-1) dry wt. cell h(-1) and 2.6 g sugar g(-1) dry wt. cell h(-1), respectively, at a dilution rate of 1.5 h(-1).

  12. Immobilized-cell-augmented activated sludge process for treating wastewater containing hazardous compounds.

    Science.gov (United States)

    Jittawattanarat, Rungrod; Kostarelos, Konstantinos; Khan, Eakalak

    2007-05-01

    A novel bioaugmentation scheme called immobilized-cell-augmented activated sludge (ICAAS) was developed. Offline enricher reactors were used to maintain immobilized acclimated cells applied to augment completely mixed activated sludge (CMAS) treating a pentachlorophenol (PCP) pulse loading. Cellulose triacetate (CA) and powder activated carbon (PAC) combined with CA (PAC + CA) were the two media types used for entrapping the PCP-degrading culture. With ICAAS at 5% by volume augmentation, PCP removal of 73.1 and 75.1% via biodegradation, volatilization, and adsorption onto suspended cells, entrapped cells, and media was achieved for the systems with CA and PAC + CA media, respectively, while PCP removal in a control CMAS, which had a comparable level of combined PCP adsorption onto suspended cells and volatilization as the ICAAS, was 48.7%. Results further showed that the immobilized cells retained their PCP-degrading ability when they were fed with the inducer (PCP) once every 20 days.

  13. Ethanol fermentation of molasses by Saccharomyces cerevisiae cells immobilized onto sugar beet pulp

    Directory of Open Access Journals (Sweden)

    Vučurović Vesna M.

    2012-01-01

    Full Text Available Natural adhesion of Saccharomyces cerevisiae onto sugar beet pulp (SBP is a very simple and cheap immobilization method for retaining high cells density in the ethanol fermentation system. In the present study, yeast cells were immobilized by adhesion onto SBP suspended in the synthetic culture media under different conditions such as: glucose concentration (100, 120 and 150 g/l, inoculum concentration (5, 10 and 15 g/l dry mass and temperature (25, 30, 35 and 40°C. In order to estimate the optimal immobilization conditions the yeast cells retention (R, after each immobilization experiment was analyzed. The highest R value of 0.486 g dry mass yeast /g dry mass SBP was obtained at 30°C, glucose concentration of 150 g/l, and inoculum concentration of 15 g/l. The yeast immobilized under these conditions was used for ethanol fermentation of sugar beet molasses containing 150.2 g/l of reducing sugar. Efficient ethanol fermentation (ethanol concentration of 70.57 g/l, fermentation efficiency 93.98% of sugar beet molasses was achieved using S. cerevisiae immobilized by natural adhesion on SBP. [Projekat Ministarstva nauke Republike Srbije, br. TR-31002

  14. Preparation of corncob grits as a carrier for immobilizing yeast cells for ethanol production.

    Science.gov (United States)

    Lee, Sang-Eun; Lee, Choon Geun; Kang, Do Hyung; Lee, Hyeon-Yong; Jung, Kyung-Hwan

    2012-12-01

    In this study, DEAE-corncobs [delignified corncob grits derivatized with 2-(diethylamino)ethyl chloride hydrochloride (DEAE·HCl)] were prepared as a carrier to immobilize yeast (Saccharomyces cerevisiae) for ethanol production. The immobilized yeast cell reactor produced ethanol under optimized DEAE·HCl derivatization and adsorption conditions between yeast cells and the DEAE-corncobs. When delignified corncob grit (3.0 g) was derivatized with 0.5M DEAE·HCl, the yeast cell suspension (OD600 = 3.0) was adsorbed at >90% of the initial cell OD600. This amount of adsorbed yeast cells was estimated to be 5.36 mg-dry cells/g-DEAE corncobs. The Qmax (the maximum cell adsorption by the carrier) of the DEAE-corncobs was estimated to be 25.1 (mg/g), based on a Languir model biosorption isotherm experiment. When we conducted a batch culture with medium recycling using the immobilized yeast cells, the yeast cells on DEAE-corncobs produced ethanol gradually, according to glucose consumption, without cells detaching from the DEAE-corncobs. We observed under electron microscopy that the yeast cells grew on the surface and in the holes of the DEAEcorncobs. In a future study, DEAE-corncobs and the immobilized yeast cell reactor system will contribute to bioethanol production from biomass hydrolysates.

  15. Continuous ethanol production from Jerusalem artichoke tubers. II. Use of immobilized cells of Kluyveromyces marxianus

    Energy Technology Data Exchange (ETDEWEB)

    Margaritis, A.; Bajpai, P.

    1982-07-01

    Kluyveromyces marxianus UCD (FST) 55-82 cells were immobilized in Na alginate beads and used in a packed-bed bioreactor system for the continuous production of ethanol from the extract of Jerusalem artichoke tubers. Volumetric ethanol productivities of 104 and 80 g ethanol/L/h were obtained at 80 and 92% sugar utilization, respectively. The maximum volumetric ethanol productivity of the immobilized cell bioreactor system was found to be 15 times higher than that of an ordinary continuous-stirred-tank (CST) bioreactor using free cells of Kluyveromyces marxianus. The immobilized cell bioreactor system was operated continuously at a constant dilution rate of 0.66/h for 12 days resulting in only an 8% loss of the original immobilized cell activity, which corresponds to an estimated half-life of ca. 72 days. The maximum specific ethanol productivity and maximum specific sugar uptake rate of the immobilized cells were found to be 0.55 g ethanol/g biomass/h and 1.21 g sugars/g biomass/h, respectively. (Refs. 27).

  16. Recycling of Immobilized Cells for Aerobic Biodegradation of Phenol in a Fluidized Bed Bioreactor

    Directory of Open Access Journals (Sweden)

    Zainab Z. Ismail

    2015-08-01

    Full Text Available Biodegradation is an environmentally friendly and cost-effective alternative that proved to be efficient for the removal of toxic phenol compounds from aqueous solutions. However, it has been reported that phenol is inhibitory to bacterial growth at concentrations above 0.05 g/L. This study was undertaken to study the degradation of phenol at initial concentrations of 20 mg/L by Bacillus cells individually immobilized in two different matrices including polyvinyl alcohol-sodium alginate (PVA-SA and polyvinyl alcohol-guar gum (PVA-GG. Results of batch experiments demonstrated that complete removal of phenol was obtained using immobilized cells in the first cycle after 270 and 300 min using cells immobilized in PVA-SA and PVA-GG. Additional cycles were conducted to evaluate the validity of recycling the beads of immobilized cells for phenol biodegradation. Results revealed that the phenol percentage removals were 96, 90, 83, and 75% for the second, third, fourth, and fifth cycles, respectively after 270 min. However, complete removal of phenol was obtained at extended time durations up to 300, 360, and 390 for the second, third, and fourth cycles, respectively. Also, the potential of immobilized cells versus free cells for the degradation of higher phenol concentration up to 50 mg/L was investigated.

  17. Biodegradation of pesticide profenofos by the free and immobilized cells of Pseudoxanthomonas suwonensis strain HNM.

    Science.gov (United States)

    Talwar, Manjunatha P; Ninnekar, Harichandra Z

    2015-09-01

    Profenofos is an organophosphate pesticide used extensively in agriculture to control pests. A bacterium capable of degrading profenofos was isolated from pesticide-contaminated soil samples and identified as Pseudoxanthomonas suwonensis strain HNM based on its morphological and biochemical characteristics and phylogenetic analysis of 16S rRNA gene sequences. 4-Bromo-2-chlorophenol was identified as a metabolite of profenofos degradation by HPLC and GC-MS analysis. The organism degraded profenofos by hydrolysis to yield 4-bromo-2-chlorophenol which was further utilized as carbon source for growth. The organism utilized various organophosphate pesticides such as temephos, quinalphos, and chloropyrifos as carbon sources. The optimum conditions for degradation of profenofos by P. suwonensis strain HMN were found to be at pH 7 and 30 °C. We have investigated the rate of degradation of profenofos by the free and immobilized cells of P. suwonensis strain HNM in various matrices such as sodium alginate (SA), sodium alginate-polyvinyl alcohol (SA-PVA), and SA-bentonite clay. The rate of degradation of 3 and 6 mM profenofos by the freely suspended cells were compared with that by immobilized cells in batches and semi-continuous with shaken cultures. The SA-bentonite clay-immobilized cells showed higher rate of degradation of 3 and 6 mM profenofos then freely suspended cells and cells immobilized in SA and SA-PVA. The SA-bentonite clay-immobilized cells of P. suwonensis strain HNM could be reused for more than 32 cycles without losing their degradation capacity. Thus, the immobilized cells are more efficient than freely suspended cells for the degradation of organophosphate pesticide contaminated water.

  18. Continuous ethanol production from Jerusalem artichokes stalks using immobilized cells of Kluyveromyces marxianus

    Energy Technology Data Exchange (ETDEWEB)

    Bajpai, P.; Margaritis, A.

    1986-01-01

    Continuous production of ethanol from the extract of Jerusalem artichoke stalks was investigated in a packed bed bioreactor using Kluyveromyces marxianus cells immobilized in calcium alginate gel beds. Maximum conversion of the sugars to ethanol was achieved with a yield of about 98% of the theoretical. Volumetric ethanol productivities of 102 grams of ethanol per litre per hour and 92 grams ethanol per liter per hour were obtained at 87% and 90% conversion respectively for an inlet substrate concentration of 100 gram sugars per liter. The maximum specific ethanol production rate and maximum specific total sugar uptake rate of the immobilized cells were found to be 0.96 gram ethanol per gram immobilized cells per hour and 2.06 gram sugars per gram immobilized cells per hour respectively. The immobilized cell bioreactor was run continuously at a dilution rate of 2.12 per hour for 30 days which resulted in a loss of 30% of the original activity. The half life of the bioreactor was estimated to be about 56 days.

  19. Investigation of interaction between the drug and cell membrane by capillary electrophoresis

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    By introducing cell membrane into electrophoretic buffer as pseudo-stationary phase,a novel capillary electrophoresis method was established to explore the interaction between drugs and cell membrane,where the interaction between citalopram and rabbit red blood cell membrane was used as an example. A series of concentrations of cell membrane were suspended into the running buffer by peak-shift method. The binding constant of citalopram to rabbit red blood cell membrane of 0.977 g-1·L was obtained after treatment of Scatchard plot. This method could provide not only a new way for the investigation on the interactions between drugs and cell membrane,but also a new approach for high throughput screening of the drug membrane permeability,biological activity,and evaluating drugs in vivo.

  20. Implementation of Microfluidic Chip Electrophoresis for the Detection of B-cell Clonality

    Directory of Open Access Journals (Sweden)

    Vazan M

    2016-04-01

    Full Text Available Introduction: A clonal population of B-cells is defined as those cells arising from the mitotic division of a single somatic cell with the same rearrangement of immunoglobulin genes. This gives rise to DNA markers for each individual lymphoid cell and its progenies and enables us to study clonality in different B-cell malignancies using multiplex polymerase chain reaction - PCR. The BIOMED-2 protocol has been implemented for clonality detection in lymphoproliferative diseases and exploits multiplex PCR reaction, subsequently analyzed by heteroduplex analysis (HDA using polyacrylamide gel electrophoresis (PAGE. With the advent of miniaturization and automation of molecular biology methods, lab-on-chip technologies were developed and replace partially the conventional approaches. We tested device for microfluidic chip, which is used for B-cells clonality analysis, using a PCR reaction for three subregions called frameworks (FR of the immunoglobulin heavy locus (IGH gene.

  1. Pancreatic cell immobilization in alginate beads produced by emulsion and internal gelation.

    Science.gov (United States)

    Hoesli, Corinne A; Raghuram, Kamini; Kiang, Roger L J; Mocinecová, Dušana; Hu, Xiaoke; Johnson, James D; Lacík, Igor; Kieffer, Timothy J; Piret, James M

    2011-02-01

    Alginate has been used to protect transplanted pancreatic islets from immune rejection and as a matrix to increase the insulin content of islet progenitor cells. The throughput of alginate bead generation by the standard extrusion and external gelation method is limited by the rate of droplet formation from nozzles. Alginate bead generation by emulsion and internal gelation is a scaleable alternative that has been used with biological molecules and microbial cells, but not mammalian cells. We describe the novel adaptation of this process to mammalian cell immobilization. After optimization, the emulsion process yielded 90 ± 2% mouse insulinoma 6 (MIN6) cell survival, similar to the extrusion process. The MIN6 cells expanded at the same rate in both bead types to form pseudo-islets with increased glucose stimulation index compared to cells in suspension. The emulsion process was suitable for primary pancreatic exocrine cell immobilization, leading to 67 ± 32 fold increased insulin expression after 10 days of immobilized culture. Due to the scaleability and broad availability of stirred mixers, the emulsion process represents an attractive option for laboratories that are not equipped with extrusion-based cell encapsulators, as well as for the production of immobilized or encapsulated cellular therapeutics on a clinical scale.

  2. Nitrilase-catalysed conversion of acrylonitrile by free and immobilized cells of Streptomyces sp.

    Indian Academy of Sciences (India)

    V K Nigam; A K Khandelwal; R K Gothwal; M K Mohan; B Choudhury; A S Vidyarthi; P Ghosh

    2009-03-01

    The biotransformation of acrylonitrile was investigated using thermophilic nitrilase produced from a new isolate Streptomyces sp. MTCC 7546 in both the free and immobilized state. Under optimal conditions, the enzyme converts nitriles to acids without the formation of amides. The whole cells of the isolate were immobilized in agar-agar and the beads so formed were evaluated for 25 cycles at 50°C. The enzyme showed a little loss of activity during reuse. Seventy-one per cent of 0.5 M acrylonitrile was converted to acid at 6 h of incubation at a very low density of immobilized cells, while 100% conversion was observed at 3 h by free cells.

  3. Surface design of antibody-immobilized thermoresponsive cell culture dishes for recovering intact cells by low-temperature treatment.

    Science.gov (United States)

    Kobayashi, Jun; Hayashi, Masaki; Ohno, Takahiro; Nishi, Masanori; Arisaka, Yoshinori; Matsubara, Yoshinori; Kakidachi, Hiroshi; Akiyama, Yoshikatsu; Yamato, Masayuki; Horii, Akihiro; Okano, Teruo

    2014-11-01

    Antibody-immobilized thermoresponsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [poly(IPAAm-co-CIPAAm)]-grafted cell culture surfaces were designed to enhance both the initial adhesion of weakly adhering cells and the ability of cells to detach in response to low temperature through the regulation of affinity binding between immobilized antibodies and antigens on the cellular surface. Ty-82 cells and neonatal normal human dermal fibroblasts (NHDFs), which express CD90 on the cell surface, adhered to anti-CD90 antibody-immobilized thermoresponsive surfaces at 37°C, a condition at which the grafted thermoresponsive polymer chains shrank. Adherent Ty-82 cells were detached from the surfaces by lowering the temperature to 20°C and applying external forces, such as pipetting, whereas cultured NHDF sheets spontaneously detached themselves from the surface in response to reduced temperature alone. When the temperature was decreased to 20°C, the swelling of grafted thermoresponsive polymer chains weakened the affinity binding between immobilized antibody and antigen on the cells due to the increasing steric hindrance of the polymer chains around the antigen-recognition site of the immobilized antibodies. No contamination was detected on cells harvested from covalently immobilized antibodies on the culture surfaces by low-temperature treatment, whereas a carryover of the antibody and avidin from the avidin-biotin binding surface was observed. Furthermore, the initial adhesion of adipose tissue-derived cells, which adhere weakly to PIPAAm-grafted surfaces, was enhanced on the antibody-immobilized thermoresponsive surfaces.

  4. The Influence of Dopants on the Effectiveness of Alginate Beads in Immobilized Cell Reactors.

    Science.gov (United States)

    Nordmeier, Akira; Chidambaram, Dev

    2016-04-01

    Zymomonas mobilis immobilized in doped calcium alginate (Ca-alginate) was successfully employed for the production of ethanol in an immobilized cell reactor. Polyethylene oxide and F127 dimethacrylate were evaluated as potential dopants for Ca-alginate beads to decrease lag time and increase initial ethanol yield. The influence of the type and concentration of the dopant on the effectiveness of the microbe immobilized in Ca-alginate beads to produce ethanol was studied, and results were compared to the widely used 2 % Ca-alginate with no dopants, which acted as control. Immobilized cell reactors that were operated using beads doped with 0.25 % polyethylene oxide (PEO) reached an ethanol yield of ∼70 % in 24 h, which was significantly higher than an ethanol yield of 25 % obtained for the control reactor operated using undoped Ca-alginate beads. This study shows that the use of water-soluble dopants can potentially reduce the lag phase and thus improve the initial production yield of immobilized cell reactors, likely due to an increase in porosity and diffusion rate of the doped beads.

  5. STUDY ON ALCOHOLIC FERMENTATION IN A STATIONARY BASKET BIOREACTOR WITH IMMOBILIZED YEAST CELLS

    Directory of Open Access Journals (Sweden)

    Dan Caşcaval

    2011-02-01

    Full Text Available The use of a stationary basket bioreactor with immobilized S. cerevisiae cells indicated the possibility to extend the number of alcoholic fermentation cycles that can be carried out with the same biocatalysts to over nine. Although the rates of glucose consumption and ethanol production were lower than those recorded for the mobile beds of immobilized yeast cells, the mechanical lysis of the biocatalysts is avoided in the case of basket bed. Due to the substrate and product accumulation inside the basket bed, the fermentation process can be improved by washing out the biocatalysts bed over two or four cycles.

  6. Immobilization of cells with nitrilase activity from a thermophilic bacterial strain.

    Science.gov (United States)

    Kabaivanova, L; Dobreva, E; Dimitrov, P; Emanuilova, E

    2005-01-01

    Cells of the moderately thermophilic Bacillus sp. UG-5B strain, producing nitrilase (EC3.5.5.1), which converts nitriles directly to the corresponding acid and ammonia, were immobilized using different types of matrices and techniques. A variety of sol-gel silica hybrids were tested for entrapment and adsorption of bacterial cells as well as chemical binding on polysulphone membranes. Activation of the matrix surface with formaldehyde led to an increase in immobilization efficiency and operational stability of the biocatalysts. Among the supports screened, membranes gave the best results for enzyme activity and especially operational stability, with retention of 100% activity after eight reaction cycles.

  7. Enzymatic production of atranorin: a component of the oak moss absolute by immobilized lichen cells.

    Science.gov (United States)

    Vicente, C; Fontaniella, B; Millanes, A M; Sebastián, B; Legaz, M E

    2003-04-01

    Cells of the lichen, Evernia prunastri, immobilized in calcium alginate were able to produce the depside atranorin from acetate. The synthesis of the depside was enhanced by molecular oxygen and NADH. This enhancement suggested the participation of an oxidase and an alcohol dehydrogenase to produce an aldehyde-substituted phenolic acid, hematommic acid, as the most probable precursor of atranorin. The participation of both enzymes was confirmed by loading immobilized cells with sodium azide, an inhibitor of several metallo-oxidases, and pyrazole, an inhibitor of alcohol dehydrogenase, which impeded atranorin production and accumulated beta-methyl orsellinate (after azide loading) or its alcohol derivative (after pirazole treatment).

  8. Recent developments in capillary and chip electrophoresis of bioparticles: Viruses, organelles, and cells.

    Science.gov (United States)

    Subirats, Xavier; Blaas, Dieter; Kenndler, Ernst

    2011-06-01

    In appropriate aqueous buffer solutions, biological particles usually exhibit a particular electric surface charge due to exposed charged or chargeable functional groups (amino acid residues, acidic carbohydrate moieties, etc.). Consequently, these bioparticles can migrate in solution under the influence of an electric field allowing separation according to their electrophoretic mobilities or their pI values. Based on these properties, electromigration methods are of eminent interest for the characterization, separation, and detection of such particles. The present review discusses the research papers published between 2008 and 2010 dealing with isoelectric focusing and zone electrophoresis of viruses, organelles and microorganisms (bacteria and yeast cells) in the capillary and the chip format.

  9. Immobilization of anode-attached microbes in a microbial fuel cell.

    KAUST Repository

    Wagner, Rachel C

    2012-01-03

    Current-generating (exoelectrogenic) bacteria in bioelectrochemical systems (BESs) may not be culturable using standard in vitro agar-plating techniques, making isolation of new microbes a challenge. More in vivo like conditions are needed where bacteria can be grown and directly isolated on an electrode. While colonies can be developed from single cells on an electrode, the cells must be immobilized after being placed on the surface. Here we present a proof-of-concept immobilization approach that allows exoelectrogenic activity of cells on an electrode based on applying a layer of latex to hold bacteria on surfaces. The effectiveness of this procedure to immobilize particles was first demonstrated using fluorescent microspheres as bacterial analogs. The latex coating was then shown to not substantially affect the exoelectrogenic activity of well-developed anode biofilms in two different systems. A single layer of airbrushed coating did not reduce the voltage produced by a biofilm in a microbial fuel cell (MFC), and more easily applied dip-and-blot coating reduced voltage by only 11% in a microbial electrolysis cell (MEC). This latex immobilization procedure will enable future testing of single cells for exoelectrogenic activity on electrodes in BESs.

  10. Immobilization of Saccharomyces cerevisiae cells and Rhizomucor miehei lipase for the production and extractive biocatalysis of ethanol

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, A.C. [Instituto Nacional de Engenharia e Tecnologia Industrial, Lisboa (Portugal). Dept. de Energias Renovaveis; Rosa, M.F. [Instituto Nacional de Engenharia e Tecnologia Industrial, Lisboa (Portugal). Dept. de Energias Renovaveis; Cabral, J.M.S. [Lab. de Engenharia Bioquimica, Centro de Engenharia Biologica e Quimica, Lisboa (Portugal); Aires-Barros, M.R. [Lab. de Engenharia Bioquimica, Centro de Engenharia Biologica e Quimica, Lisboa (Portugal)

    1997-05-01

    The production of ethanol by Saccharomyces cerevisiae immobilized cells and its esterification with oleic acid, catalysed by a lipase from Rhizomucor miehei, was the biochemical process considered as model to illustrate the concept of extractive biocatalysis. The selection of the most suitable support for lipase immobilization was carried out. The best results for the ethanol/oleic acid esterification reaction were obtained with the lipase adsorbed on a polyamide type support, Accurel EP 700. The immobilization method was optimized in terms of immobilization pH, contact time and protein/support ratio. The better performances of the extractive fermentations of ethanol were obtained when entrapped k-carrageenan Saccharomyces cerevisiae cells and a lipase from Rhizomucor miehei, free or immobilized in Accurel EP 700, were used simultaneously. The observed reutilization capacity of the immobilized enzyme could be advantageous for its application in a continuous reactor. (orig.). With 5 figs., 2 tabs.

  11. Polygalacturonase production by calcium alginate immobilized Enterobacter aerogenes NBO2 cells.

    Science.gov (United States)

    Darah, I; Nisha, M; Lim, Sheh-Hong

    2015-03-01

    Bacterial cells of Enterobacter aerogenes NBO2 were entrapped in calcium alginate beads in order to enhance polygalacturonase production compared to free cells. The optimized condition of 5 % (w/v) sodium alginate concentration, agitation speed of 250 rpm, and 15 beads of calcium alginate with inoculum size of 4 % (v/v; 5.4 × 10(7) cells/ml) produced 23.48 U/mL of polygalacturonase compared to free cells of 18.54 U/ml. There was about 26.6 % increment in polygalaturonase production. However, in this study, there was 296.6 % of increment in polygalacturonase production after improvement parameters compared to before improvement parameters of calcium alginate bead immobilization cells (5.92 U/ml). This research has indicated that optimized physical parameters of calcium alginate bead immobilization cells have significantly enhanced the production of polygalacturonase.

  12. Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin Kyu

    2007-10-15

    Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals. Recently, the importance of ionizing radiation and chemicals has been recognized since radio- and chemical therapy is directly related to the control of various diseases such as cancer. Radiation and the chemicals can cause biological damages while they have great applicability. It is of necessity to analyze rapidly, easily and accurately the biological effects, especially DNA damage due to those factors. Recently SCGE (single cell gel electrophoresis assay, alias comet assay) has been developed for the efficient evaluation of DNA damage. In this report, the comprehensive review will be given on the rationale, the technical applications and the advantages and shortcomings of SCGE assay. This method can be directly applied to study on toxicity, cancer, and aging in terms of the evaluation of DNA damages due to radiation and chemicals on human cellular level. It is also suggested that comet assay be used for testing genotoxicity of suspected substances, detecting irradiated foods, screening radioprotective candidates, and studying DNA repair process in various biological systems.

  13. Effect of immobilized cells in calcium alginate beads in alcoholic fermentation.

    Science.gov (United States)

    Duarte, Juliana C; Rodrigues, J Augusto R; Moran, Paulo J S; Valença, Gustavo P; Nunhez, José R

    2013-05-30

    Saccharomyces cerevisiae cells were immobilized in calcium alginate and chitosan-covered calcium alginate beads and studied in the fermentation of glucose and sucrose for ethanol production. The batch fermentations were carried out in an orbital shaker and assessed by monitoring the concentration of substrate and product with HPLC. Cell immobilization in calcium alginate beads and chitosan-covered calcium alginate beads allowed reuse of the beads in eight sequential fermentation cycles of 10 h each. The final concentration of ethanol using free cells was 40 g L-1 and the yields using glucose and sucrose as carbon sources were 78% and 74.3%, respectively. For immobilized cells in calcium alginate beads, the final ethanol concentration from glucose was 32.9 ± 1.7 g L-1 with a 64.5 ± 3.4% yield, while the final ethanol concentration from sucrose was 33.5 ± 4.6 g L-1 with a 64.5 ± 8.6% yield. For immobilized cells in chitosan-covered calcium alginate beads, the ethanol concentration from glucose was 30.7 ± 1.4 g L-1 with a 61.1 ± 2.8% yield, while the final ethanol concentration from sucrose was 31.8 ± 6.9 g L-1 with a 62.1 ± 12.8% yield. The immobilized cells allowed eight 10 h sequential reuse cycles to be carried out with stable final ethanol concentrations. In addition, there was no need to use antibiotics and no contamination was observed. After the eighth cycle, there was a significant rupture of the beads making them inappropriate for reuse.

  14. Use of Saccharomyces cerevisiae cells immobilized on orange peel as biocatalyst for alcoholic fermentation

    Energy Technology Data Exchange (ETDEWEB)

    Plessas, S.; Bekatorou, A; Koutinas, A.A.; Soupioni, M. [University of Patras (Greece). Department of Chemistry, Food Biotechnology Group; Banat, I.M.; Marchant, R. [University of Ulster, Coleraine, N. Ireland (United Kingdom). School of Biomedical Sciences

    2007-03-15

    A biocatalyst was prepared by immobilizing a commercial Saccharomyces cerevisiae strain (baker's yeast) on orange peel pieces for use in alcoholic fermentation and for fermented food applications. Cell immobilization was shown by electron microscopy and by the efficiency of the immobilized biocatalyst for alcoholic fermentation of various carbohydrate substrates (glucose, molasses, raisin extracts) and at various temperatures (30-15 {sup o}C). Fermentation times in all cases were low (5-15 h) and ethanol productivities were high (av. 150.6 g/ld) showing good operational stability of the biocatalyst and suitability for commercial applications. Reasonable amounts of volatile by-products were produced at all temperatures studied, revealing potential application of the proposed biocatalyst in fermented food applications, to improve productivities and quality. (author)

  15. Adlayer-mediated antibody immobilization to stainless steel for potential application to endothelial progenitor cell capture.

    Science.gov (United States)

    Benvenuto, Pasquale; Neves, Miguel A D; Blaszykowski, Christophe; Romaschin, Alexander; Chung, Timothy; Kim, Sa Rang; Thompson, Michael

    2015-05-19

    This work describes the straightforward surface modification of 316L stainless steel with BTS, S-(11-trichlorosilylundecanyl)-benzenethiosulfonate, a thiol-reactive trichlorosilane cross-linker molecule designed to form intermediary coatings with subsequent biofunctionalization capability. The strategy is more specifically exemplified with the immobilization of intact antibodies and their Fab' fragments. Both surface derivatization steps are thoroughly characterized by means of X-ray photoelectron spectroscopy. The antigen binding capability of both types of biofunctionalized surfaces is subsequently assessed by fluorescence microscopy. It was determined that BTS adlayers achieve robust immobilization of both intact and fragmented antibodies, while preserving antigen binding activity. Another key finding was the observation that the Fab' fragment immobilization strategy would constitute a preferential option over that involving intact antibodies in the context of in vivo capture of endothelial progenitor cells in stent applications.

  16. Removal of Cadmium and Zinc from Soil using Immobilized Cell of Biosurfactant Producing Bacteria

    Directory of Open Access Journals (Sweden)

    Charoon Sarin

    2010-07-01

    Full Text Available Immobilized biosurfactant producing bacteria (Bacillus subtilis TP8 and Pseudomonas fluorescens G7 were assessed for survival in heavy metal contaminated soil and for their ability to remove cadmium and zinc from contaminated soil. P. fluorescens G7 was considered to be a good candidate for bioremediation of heavy metals because of its high minimum inhibitory concentrations (MIC for each heavy metal and because of the obviously increased numbers of cell surviving after incubation in the heavy metal contaminated soil up to 4 weeks. The results of soil remediation showed that approximately 19% of Zn and 16.7% of Cd could be removed by this immobilized biosurfactant producing bacteria after incubation for 2 weeks. The results confirm the potential applicability of the immobilized biosurfactant producing bacteria for heavy metal bioremediation.

  17. Immobilization of Biocatalysts and Cells on Hybrid Membranes Syntheses on Sol-gel Method

    Directory of Open Access Journals (Sweden)

    Yotova L.

    2007-12-01

    Full Text Available The investigations in the area of enzyme action in the living organisms give us the opportunity for applications of these biochemical catalysts in the different purposes of medicine, industry and analytical practice. Using of soluble enzymes is connected with many difficulties, because the enzymes are no regenerative and they are instability. By immobilization of the enzymes on to different carriers the more of these problems are overcome. The application of soluble enzymes caused many difficulties, by the reason that the enzymes can not be regenerated. The immobilization of the enzymes by means of different reagents and carriers overcomes this problem. The aim of this study to develop a sol-gel method of synthesis of new hybrid membrane, with immobilized biocatalysts (microbial cells and enzymes for biosensor construction. This study shows that hybrid organic-inorganic membranes were synthesized.

  18. Chitin hydrolysis assisted by cell wall degrading enzymes immobilized of Thichoderma asperellum on totally cinnamoylated D-sorbitol beads

    Energy Technology Data Exchange (ETDEWEB)

    Fernandes, Kátia F., E-mail: katia@icb.ufg.br [Departamento de Bioquímica e Biologia Molecular, Instituo de Ciências Biológicas, Universidade Federal de Goiás, Cx. Postal 131, 74001-970 Goiânia, GO (Brazil); Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain); Cortijo-Triviño, David [Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain); Batista, Karla A.; Ulhoa, Cirano J. [Departamento de Bioquímica e Biologia Molecular, Instituo de Ciências Biológicas, Universidade Federal de Goiás, Cx. Postal 131, 74001-970 Goiânia, GO (Brazil); García-Ruiz, Pedro A. [Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain)

    2013-07-01

    In this study, cell wall degrading enzymes produced by Thrichoderma asperellum (TCWDE) were immobilized on totally cinnamoylated D-sorbitol (TCNSO) beads and used for chitin hydrolysis. In order to optimize immobilization efficiency, the reaction time was varied from 2 to 12 h and reactions were conducted in the presence or absence of Na{sub 2}SO{sub 4}. Immobilized enzymes were analysed concerning to thermal and operational stability. Immobilization in presence of Na{sub 2}SO{sub 4} was 54% more efficient than immobilization in absence of salt. After optimization, 32% of the total enzyme offered was immobilized, with 100% of bounding efficiency, measured as the relation between protein and enzyme immobilized. Free and TCNSO–TCWDE presented very similar kinetics with maximum hydrolysis reached at 90 min of reaction. Thermal stability of both free and TCNSO–TCWDE was similar, with losses in activity after 55 °C. Moreover, free and TCNSO–TCWDE retained 100% activity after 3 h incubation at 55 °C. TCNSO–TCWDE were used in a bath-wise reactor during 14 cycles, producing 1825 μg of N-acetylglucosamine (NAG) maintaining 83% of initial activity. - Highlights: • TCWDE immobilized on TCNSO, a support with highly hydrophobic character • New immobilization strategy for immobilization on a hydrophobic support • TCNSO–TCWDE were retained during washes and during incubation at 55 °C for 3 h.

  19. Electrochemical Detection of Alkaline Phosphatase in BALB/c Mouse Fetal Liver Stromal Cells with Capillary Electrophoresis

    Institute of Scientific and Technical Information of China (English)

    Xue Mei SUN; Dong LI; Zeng Liang BAI; Wen Rui JIN

    2004-01-01

    A method for determination of alkaline phosphatase (ALP) in BALB/c mouse fetal liver stromal cells has been described based on the catalytic reaction. After the cell extract is incubated with the substrate disodium phenyl phosphate, the reaction product phenol generated by ALP is determined by capillary electrophoresis with electrochemical detection.

  20. Gibberellic acid production by free and immobilized cells in different culture systems.

    Science.gov (United States)

    Durán-Páramo, Enrique; Molina-Jiménez, Héctor; Brito-Arias, Marco A; Robles-Martínez, Fabián

    2004-01-01

    Gibberellic acid production was studied in different fermentation systems. Free and immobilized cells of Gibberella fujikuroi cultures in shake-flask, stirred and fixed-bed reactors were evaluated for the production of gibberellic acid (GA3). Gibberellic acid production with free cells cultured in a stirred reactor reached 0.206 g/L and a yield of 0.078 g of GA3/g biomass.

  1. Analysis of Differential Gel Electrophoresis of Paclitaxol Resistant and Sensitive Lung Adenocarcinoma Cells' Secretome

    Directory of Open Access Journals (Sweden)

    Tianqing CHU

    2009-07-01

    Full Text Available Background and objective Paclitaxol (PTX resistance is one of main factors which affect the outcome of chemotherapy of lung adenocarcinoma. The aim of this study is to compare the secreted protein expression profiles between Paclitaxol (PTX resistant and sensitive lung adenocarcinoma cells by proteomic research method, so as to provide evidence of choosing individual chemotherapy drugs in clinical treatment. Methods Total secreted proteins extracted from a PTX sensitive cell line A549 and a PTX resistant cell line A549-Taxol were separated by fluorscent differential gel electrophoresis (DIGE. High quality 2-DE profiles were obtained and analyzed by Decyder 6.5 analysis software to screen differentially expressed protein spots. Those spots were identified by mass spectrometry. Results 2-DE patterns of lung adenocarcinoma cells with high-resolution and reproducibility were obtained. 76 significantly differentially expressed protein spots were screened, 19 proteins were identified by mass spectrometry. The identified proteins could be classified into different catogories: metabolic enzyme, extracellular matrix (ECM degradation enzyme, cytokine, signal transducer, cell adhesion, and so on. Conclusion Multiple secreted proteins related to chemoresistance of A549-Taxol cells were identified in this study for the first time. The results presented here would provide clues to identify new serologic chemoresistant biomarkers of NSCLC.

  2. Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two-dimensional gel electrophoresis patterns based on mass spectrometric protein identification

    DEFF Research Database (Denmark)

    Nawrocki, A; Larsen, Martin Røssel; Podtelejnikov, A V;

    1998-01-01

    Separation of proteins on either carrier ampholyte-based or immobilized pH gradient-based two-dimensional (2-D) gels gives rise to electrophoretic patterns that are difficult to compare visually. In this paper we have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI......-MS) to determine the identities of 335 protein spots in these two 2-D gel systems, including a substantial number of basic proteins which had never been identified before. Proteins that were identified in both gel systems allowed us to cross-reference the gel patterns. Vector analysis of these cross...

  3. A response calculus for immobilized T cell receptor ligands

    DEFF Research Database (Denmark)

    Andersen, P S; Menné, C; Mariuzza, R A

    2001-01-01

    To address the molecular mechanism of T cell receptor (TCR) signaling, we have formulated a model for T cell activation, termed the 2D-affinity model, in which the density of TCR on the T cell surface, the density of ligand on the presenting surface, and their corresponding two-dimensional affini...

  4. Monitoring exocytosis and release from individual mast cells by capillary electrophoresis and UV imaging microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Yeung, E.S. [Ames Lab., IA (United States)]|[Iowa State Univ., Ames, IA (United States); Lillard, S.J. [Stanford Univ., CA (United States); McCloskey, M.A. [Iowa State Univ., Ames, IA (United States)

    1997-12-31

    The complex temporal evolution of on-column exocytotic release of serotonin from individual peritoneal mast cells (RPMCs) was monitored by using capillary electrophoresis and UV imaging microscopy. Laser-induced native fluorescence detection with 275-nm excitation was used, and a detection limit of 1.7 amol (S/N = 3; rms) was obtained for serotonin. A physiological running buffer was used to ensure that the cell remained viable throughout. The secretagogue was polymyxin B sulfate (Pmx). Following the injection of a single mast cell into the capillary, electromigration of Pmx toward and past the cell induced degranulation and release of serotonin. The time course of release was registered in the electropherograms with subsecond resolution. Subsequent introduction of SDS caused the cell to lyse completely and allowed the residual serotonin to be quantified. The average amount of serotonin observed per RPMC was 1.6 {+-} 0.6 fmol; the average percentage of serotonin released was 28 {+-} 14%. Events that are consistent with released serontonin from single submicron granules (250 aL each) were evident, each of which contained an average amount of 5.9 {+-} 3 amol. Alternatively, UV movies can be taken of the entire event to provide temporal and spatial information.

  5. Optimizing Immobilized Enzyme Performance in Cell-Free Environments to Produce Liquid Fuels

    Energy Technology Data Exchange (ETDEWEB)

    Belfort, Georges [Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering; Grimaldi, Joseph J. [Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering

    2015-01-27

    Limitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (≤0.2 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, an alternate simplified biofuel production scheme was tested based on a cell-free immobilized enzyme system. Using this cell free system, we were able to obtain about 2.6 times higher concentrations of iso-butanol using our non-optimized system as compared with live cell systems. This process involved two steps: (i) converts acid to aldehyde using keto-acid decarboxylase (KdcA), and (ii) produces alcohol from aldehyde using alcohol dehydrogenase (ADH) with a cofactor (NADH) conversion from inexpensive formate using a third enzyme, formate dehydrogenase (FDH). To increase stability and conversion efficiency with easy separations, the first two enzymes were immobilized onto methacrylate resin. Fusion proteins of labile KdcA (fKdcA) were expressed to stabilize the covalently immobilized KdcA. Covalently immobilized ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles without fusions. High conversion rates and low protein leaching were achieved by covalent immobilization of enzymes on methacrylate resin. The complete reaction scheme was demonstrated by immobilizing both ADH and fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.5 % (v/v). Further increases in titer will require continuous removal of the isobutanol using our novel brush membrane system that exhibits a 1.5 fold increase in the separation factor of isobutanol from water versus that obtained for commercial silicone rubber membranes. These bio-inspired brush membranes are based on the

  6. New method for selection of hydrogen peroxide adapted bifidobacteria cells using continuous culture and immobilized cell technology

    Directory of Open Access Journals (Sweden)

    Meile Leo

    2010-07-01

    Full Text Available Abstract Background Oxidative stress can severely compromise viability of bifidobacteria. Exposure of Bifidobacterium cells to oxygen causes accumulation of reactive oxygen species, mainly hydrogen peroxide, leading to cell death. In this study, we tested the suitability of continuous culture under increasing selective pressure combined with immobilized cell technology for the selection of hydrogen peroxide adapted Bifidobacterium cells. Cells of B. longum NCC2705 were immobilized in gellan-xanthan gum gel beads and used to continuously ferment MRS medium containing increasing concentration of H2O2 from 0 to 130 ppm. Results At the beginning of the culture, high cell density of 1013 CFU per litre of reactor was tested. The continuous culture gradually adapted to increasing H2O2 concentrations. However, after increasing the H2O2 concentration to 130 ppm the OD of the culture decreased to 0. Full wash out was prevented by the immobilization of the cells in gel matrix. Hence after stopping the stress, it was possible to re-grow the cells that survived the highest lethal dose of H2O2 and to select two adapted colonies (HPR1 and HPR2 after plating of the culture effluent. In contrast to HPR1, HPR2 showed stable characteristics over at least 70 generations and exhibited also higher tolerance to O2 than non adapted wild type cells. Preliminary characterization of HPR2 was carried out by global genome expression profile analysis. Two genes coding for a protein with unknown function and possessing trans-membrane domains and an ABC-type transporter protein were overexpressed in HPR2 cells compared to wild type cells. Conclusions Our study showed that continuous culture with cell immobilization is a valid approach for selecting cells adapted to hydrogen peroxide. Elucidation of H2O2 adaptation mechanisms in HPR2 could be helpful to develop oxygen resistant bifidobacteria.

  7. Internalization: acute apoptosis of breast cancer cells using herceptin-immobilized gold nanoparticles

    Directory of Open Access Journals (Sweden)

    Rathinaraj P

    2015-02-01

    Full Text Available Pierson Rathinaraj,1 Ahmed M Al-Jumaily,1 Do Sung Huh21Institute of Biomedical Technologies, Auckland University of Technology, Auckland, New Zealand; 2Department of Nano science and Engineering, Inje University, Gimhea, South KoreaAbstract: Herceptin, the monoclonal antibody, was successfully immobilized on gold nanoparticles (GNPs to improve their precise interactions with breast cancer cells (SK-BR3. The mean size of the GNPs (29 nm, as determined by dynamic light scattering, enlarged to 82 nm after herceptin immobilization. The in vitro cell culture experiment indicated that human skin cells (FB proliferated well in the presence of herceptin-conjugated GNP (GNP–Her, while most of the breast cancer cells (SK-BR3 had died. To elucidate the mechanism of cell death, the interaction of breast cancer cells with GNP–Her was tracked by confocal laser scanning microscopy. Consequently, GNP–Her was found to be bound precisely to the membrane of the breast cancer cell, which became almost saturated after 6 hours incubation. This shows that the progression signal of SK-BR3 cells is retarded completely by the precise binding of antibody to the human epidermal growth factor receptor 2 receptor of the breast cancer cell membrane, causing cell death.Keywords: herceptin, gold nanoparticles, SK-BR3 cells, intracellular uptake

  8. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Ozcan;

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...... at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused...

  9. Sequestration of Reactive Blue 4 by free and immobilized Bacillus subtilis cells and its extracellular polysaccharides.

    Science.gov (United States)

    Binupriya, Arthur Raj; Sathishkumar, Muthuswamy; Ku, Chang Sub; Yun, Soon-Il

    2010-03-01

    Bacillus subtilis a gram positive bacteria and its extracellular polysaccharide were used in free form as well as immobilized form as biosorbent for sequestration of an anionic dye, Reactive Blue 4 (RB) in aqueous phase. The dye uptake enhanced with decrease in pH. Extracellular polymeric substances (EPS) and free cells were found to be better adsorbents when compared to alginate immobilized cells (IC) and EPS (IEPS). The presence of functional groups in free cells and EPS was confirmed by FT-IR analysis. Immobilization resulted in poor adsorption performance due to increase in mass transfer resistance by the polymeric matrix. High Q(max) and b values were noted in the case of free cells and free EPS in contrast to IC and IEPS. From the kinetic experiments, the adsorption system was found to be a pseudo-first-order reaction at low dye concentration. Desorption of RB was found to be 100% in 1N NaOH. However, the alginate beads were found to be unstable under high alkaline conditions of NaOH.

  10. Immobilization of microalgae cells in alginate facilitates isolation of DNA and RNA.

    Science.gov (United States)

    Lopez, Blanca R; Hernandez, Juan-Pablo; Bashan, Yoav; de-Bashan, Luz E

    2017-04-01

    Isolation of nucleic acids from Chlorella is difficult, given the chemically complex nature of their cell walls and variable production of metabolites. Immobilization of microalgae in polymers adds additional difficulty. Here, we modified, amended, and standardized methods for isolation of nucleic acids and compared the yield of DNA and RNA from free-living and encapsulated microalgae C. sorokiniana. Isolation of nucleic acids from immobilized cells required two steps in dissolving the alginate matrix, releasing the cells, and mechanical disruption with glass beads. For DNA extraction, we used modified versions of a commercial kit along with the hexadecyltrimethylammonium bromide (CTAB) method. For RNA extraction, we used the commercial TRI reagent procedure and the CTAB-dithiotreitol method. Quantity and quality of nucleic acids in extracts varied with growth conditions, isolation procedures, and time of incubation of the original culture. There were consistently higher amounts of DNA and RNA in extracts from immobilized cells. Quantitatively, the modified procedure with the commercial Promega kit was the most reliable procedure for isolating DNA and a modified commercial TRI reagent procedure was the choice for isolating RNA. All four procedures eliminated proteins efficiently and had low levels of contamination from residual polysaccharides from the matrices and/or metabolites naturally produced by the microalgae. All DNA extracts under both growth conditions, time of incubation, and two isolation methods successfully amplified the 18S ribosomal RNA by PCR and quantitative reverse transcription (RT-qPCR).

  11. Measurement of oxidatively-induced clustered DNA lesions using a novel adaptation of single cell gel electrophoresis (comet assay).

    Science.gov (United States)

    Georgakilas, Alexandros G; Holt, Stewart M; Hair, Jessica M; Loftin, Charles W

    2010-12-01

    The two basic groups of complex DNA damage are double-strand breaks (DSBs) and non-DSB oxidatively-induced clustered DNA lesions (OCDLs). The single-cell gel electrophoresis (SCGE) or comet assay has been widely used for the detection of low levels of various types of DNA lesions including single-strand breaks (SSBs), DSBs, and oxidized bases per individual cell. There are limited data on the use of the comet assay for the detection of non-DSB clustered DNA lesions using different repair enzymes as enzymatic probes. This unit discusses a novel adaptation of the comet assay used to measure these unique types of lesions. Until now OCDL yields have been measured using primarily pulsed-field agarose gel electrophoresis. The advantages offered by the current approach are: (1) measurement of OCDL levels per individual cell; (2) use of a small number of cells (∼10,000) and relatively low doses of ionizing radiation (1 to 2 Gy) or low levels of oxidative stress, which are not compatible with standard agarose gel electrophoresis; and finally, (3) the assay is fast and allows direct comparison with pulsed-field gel electrophoresis results.

  12. A novel cell weighing method based on the minimum immobilization pressure for biological applications

    Science.gov (United States)

    Zhao, Qili; Shirinzadeh, Bijan; Cui, Maosheng; Sun, Mingzhu; Liu, Yaowei; Zhao, Xin

    2015-07-01

    A novel weighing method for cells with spherical and other regular shapes is proposed in this paper. In this method, the relationship between the cell mass and the minimum aspiration pressure to immobilize the cell (referred to as minimum immobilization pressure) is derived for the first time according to static theory. Based on this relationship, a robotic cell weighing process is established using a traditional micro-injection system. Experimental results on porcine oocytes demonstrate that the proposed method is able to weigh cells at an average speed of 16.3 s/cell and with a success rate of more than 90%. The derived cell mass and density are in accordance with those reported in other published results. The experimental results also demonstrated that this method is able to detect less than 1% variation of the porcine oocyte mass quantitatively. It can be conducted by a pair of traditional micropipettes and a commercial pneumatic micro-injection system, and is expected to perform robotic operation on batch cells. At present, the minimum resolution of the proposed method for measuring the cell mass can be 1.25 × 10-15 kg. Above advantages make it very appropriate for quantifying the amount of the materials injected into or moved out of the cells in the biological applications, such as nuclear enucleations and embryo microinjections.

  13. A novel cell weighing method based on the minimum immobilization pressure for biological applications

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Qili [Robotics and Mechatronics Research Laboratory, Department of Mechanical and Aerospace Engineering, Monash University, Clayton 3800 (Australia); Institute of Robotics and Automatic Information System, Nankai University, Tianjin 300071 (China); Shirinzadeh, Bijan [Robotics and Mechatronics Research Laboratory, Department of Mechanical and Aerospace Engineering, Monash University, Clayton 3800 (Australia); Cui, Maosheng [Biotechnology Lab of Animal Reproduction, Tianjin Animal Sciences, Tianjin 300112 (China); Sun, Mingzhu; Liu, Yaowei; Zhao, Xin, E-mail: zhaoxin@nankai.edu.cn [Institute of Robotics and Automatic Information System, Nankai University, Tianjin 300071 (China)

    2015-07-28

    A novel weighing method for cells with spherical and other regular shapes is proposed in this paper. In this method, the relationship between the cell mass and the minimum aspiration pressure to immobilize the cell (referred to as minimum immobilization pressure) is derived for the first time according to static theory. Based on this relationship, a robotic cell weighing process is established using a traditional micro-injection system. Experimental results on porcine oocytes demonstrate that the proposed method is able to weigh cells at an average speed of 16.3 s/cell and with a success rate of more than 90%. The derived cell mass and density are in accordance with those reported in other published results. The experimental results also demonstrated that this method is able to detect less than 1% variation of the porcine oocyte mass quantitatively. It can be conducted by a pair of traditional micropipettes and a commercial pneumatic micro-injection system, and is expected to perform robotic operation on batch cells. At present, the minimum resolution of the proposed method for measuring the cell mass can be 1.25 × 10{sup −15 }kg. Above advantages make it very appropriate for quantifying the amount of the materials injected into or moved out of the cells in the biological applications, such as nuclear enucleations and embryo microinjections.

  14. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Özcan;

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... proceeded via anthraquinone photochemistry to introduce amine functionalities at the surface followed by coupling of IL-4 through a bifunctional amine-reactive linker. X-ray photoelectron spectroscopy showed that undesirable multilayer formation of the photoactive compound could be avoided by reaction...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...

  15. Immobilization of RGD Peptides onto Decellularized Valve Scaffolds to Promote Cell Adhesion

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Porcine aortic valves were decellularized with trypsinase/EDTA and Triton-100. With the help of a coupling reagent Sulfo-LC-SPDP, the biological valve scaffolds were immobilized with one of RGD(arginine-glycine-aspartic acid) containing peptides, called GRGDSPC peptide. Myofibroblasts harvested from rats were seeded onto them. Based on the spectra of X-ray photoelectron spectroscopy, we could find conjugation of GRGDSPC peptide and the scaffolds. Cell count by both microscopy and MTT assay showed that myofibroblasts were easier to adhere to the modified scaffolds. It is proved that it is feasible to immobilize RGD peptides onto decellularized valve scaffolds, and effective to promote cell adhesion, which is beneficial for constructing tissue engineering heart valves in vitro.

  16. Continuous Ethanol Production Using Immobilized-Cell/Enzyme Biocatalysts in Fluidized-Bed Bioreactor (FBR)

    Energy Technology Data Exchange (ETDEWEB)

    Nghiem, NP

    2003-11-16

    The immobilized-cell fluidized-bed bioreactor (FBR) was developed at Oak Ridge National Laboratory (ORNL). Previous studies at ORNL using immobilized Zymomonas mobilis in FBR at both laboratory and demonstration scale (4-in-ID by 20-ft-tall) have shown that the system was more than 50 times as productive as industrial benchmarks (batch and fed-batch free cell fermentations for ethanol production from glucose). Economic analysis showed that a continuous process employing the FBR technology to produce ethanol from corn-derived glucose would offer savings of three to six cents per gallon of ethanol compared to a typical batch process. The application of the FBR technology for ethanol production was extended to investigate more complex feedstocks, which included starch and lignocellulosic-derived mixed sugars. Economic analysis and mathematical modeling of the reactor were included in the investigation. This report summarizes the results of these extensive studies.

  17. Production of biofuels from pretreated microalgae biomass by anaerobic fermentation with immobilized Clostridium acetobutylicum cells.

    Science.gov (United States)

    Efremenko, E N; Nikolskaya, A B; Lyagin, I V; Senko, O V; Makhlis, T A; Stepanov, N A; Maslova, O V; Mamedova, F; Varfolomeev, S D

    2012-06-01

    The purpose of this work was to study the possible use of pretreated biomass of various microalgae and cyanobacteria as substrates for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum cells immobilized into poly(vinyl alcohol) cryogel. To this end, the biochemical composition of photosynthetic microorganisms cultivated under various conditions was studied. The most efficient technique for pretreating microalgal biomass for its subsequent conversion into biofuels appeared to be thermal decomposition at 108 °C. For the first time the maximum productivity of the ABE fermentation in terms of hydrogen (8.5 mmol/L medium/day) was obtained using pretreated biomass of Nannochloropsis sp. Maximum yields of butanol and ethanol were observed with Arthrospira platensis biomass used as the substrate. Immobilized Clostridium cells were demonstrated to be suitable for multiple reuses (for a minimum of five cycles) in ABE fermentation for producing biofuels from pretreated microalgal biomass.

  18. Removal of Cadmium and Zinc from Soil using Immobilized Cell of Biosurfactant Producing Bacteria

    OpenAIRE

    Charoon Sarin; Siripun Sarin

    2010-01-01

    Immobilized biosurfactant producing bacteria (Bacillus subtilis TP8 and Pseudomonas fluorescens G7) were assessed for survival in heavy metal contaminated soil and for their ability to remove cadmium and zinc from contaminated soil. P. fluorescens G7 was considered to be a good candidate for bioremediation of heavy metals because of its high minimum inhibitory concentrations (MIC) for each heavy metal and because of the obviously increased numbers of cell surviving after incubation in the hea...

  19. Enhanced degradation of pendimethalin by immobilized cells of Bacillus lehensis XJU

    OpenAIRE

    More, Veena S.; Tallur, Preeti N.; Niyonzima, Francois N.; More, Sunil S.

    2015-01-01

    A bacterium capable of degrading pendimethalin was isolated from the contaminated soil samples and identified as Bacillus lehensis XJU based on 16S rRNA gene sequence analysis. 6-Aminopendimethalin and 3,4-dimethyl 2,6-dinitroaniline were identified as the metabolites of pendimethalin degradation by the bacterium. The biodegradation of pendimethalin by freely suspended and the immobilized cells of B. lehensis on various matrices namely agar, alginate, polyacrylamide, and polyurethane foam was...

  20. Application in the Ethanol Fermentation of Immobilized Yeast Cells in Matrix of Alginate/Magnetic Nanoparticles, on Chitosan-Magnetite Microparticles and Cellulose-coated Magnetic Nanoparticles

    CERN Document Server

    Ivanova, Viara; Hristov, Jordan

    2011-01-01

    Saccharomyces cerevisiae cells were entrapped in matrix of alginate and magnetic nanoparticles and covalently immobilized on magnetite-containing chitosan and cellulose-coated magnetic nanoparticles. Cellulose-coated magnetic nanoparticles with covalently immobilized thermostable {\\alpha}-amylase and chitosan particles with immobilized glucoamylase were also prepared. The immobilized cells and enzymes were applied in column reactors - 1/for simultaneous corn starch saccharification with the immobilized glucoamylase and production of ethanol with the entrapped or covalently immobilized yeast cells, 2/ for separate ethanol fermentation of the starch hydrolysates with the fixed yeasts. Hydrolysis of corn starch with the immobilized {\\alpha}-amylase and glucoamylase, and separate hydrolysis with the immobilized {\\alpha}-amylase were also examined. In the first reactor the ethanol yield reached approx. 91% of the theoretical; the yield was approx. 86% in the second. The ethanol fermentation was affected by the typ...

  1. Efficacy of whey protein gel networks as potential viability-enhancing scaffolds for cell immobilization of Lactobacillus rhamnosus GG.

    Science.gov (United States)

    Doherty, S B; Gee, V L; Ross, R P; Stanton, C; Fitzgerald, G F; Brodkorb, A

    2010-03-01

    This study investigated cell immobilization of Lactobacillus rhamnosus GG in three separate protein products: native, denatured and hydrolysed whey protein isolate (WPI). Treatments were assessed for their ability to enhance probiotic survival during storage, heat stress and ex vivo gastric incubation. Spatial distribution of probiotic cells within immobilized treatments was evaluated by atomic force and confocal scanning laser microscopy, while cell viability was enumerated by plate count and flow cytometry (FACS). Microscopic analysis of denatured treatments revealed an oasis of immobilized cells, phase-separated from the surrounding protein matrix; an environmental characteristic analogous to hydrolysed networks. Cell immobilization in hydrolysed and denatured WPI enhanced survival by 6.1+/-0.1 and 5.8+/-0.1 log10 cycles, respectively, following 14 day storage at 37 degrees C and both treatments generated thermal protection at 57 degrees C (7.3+/-0.1 and 6.5+/-0.1 log(10) cfu/ml). Furthermore, denatured WPI enhanced probiotic protection (8.9+/-0.2 log(10) cfu/ml) following 3h gastric incubation at 37 degrees C. In conclusion, hydrolysed or denatured WPI were the most suitable matrices for cell immobilization, while native protein provided the weakest safeguard against thermal and acid stress, thus making it possible to envision whey protein gel networks as protective substrates for cell immobilization applications.

  2. Enzymatic Synthesis of Agmatine by Immobilized Escherichia coli Cells with Arginine Decarboxylase Activity

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wei-guo; ZHAO Gen-hai; LIU Jun-zhong; LIU Qian; JIAO Qing-cai

    2011-01-01

    A new method for the enzymatic synthesis of agmatine by immobilized Escherichia coli cells with arginine decarboxylase(ADC)activity was established and a series of optimal reaction conditions was set down.The arginine decarboxylase showed the maximum activity when the pyridoxal phosphate(PLP)concentration was 50 mmol/L,pH=7 and 45 ℃.The arginine decarboxylase exhibited the maximum production efficiency when the substrate concentration was 100 mmol/L and the reaction time was 15 h.It was also observed that the appropriate concentration of Mg2+,especially at 0.5 mmol/L promoted the arginine decarboxylase activity; Mn2+ had little effect on the arginine decarboxylase activity.The inhibition of Cu2+ and Zn2+ to the arginine decarboxylase activity was significant.The immobilized cells were continuously used 6 times and the average conversion rate during the six-time usage was 55.6%.The immobilized cells exhibited favourable operational stability.After optimization,the maximally cumulative amount of agmatine could be up to 20 g/L.In addition,this method can also catalyze D,L-arginine to agmatine,leaving the pure optically D-arginine simultaneously.The method has a very important guiding significance to the enzymatic preparation of agmatine.

  3. Raspberry wine fermentation with suspended and immobilized yeast cells of two strains of Saccharomyces cerevisiae.

    Science.gov (United States)

    Djordjević, Radovan; Gibson, Brian; Sandell, Mari; de Billerbeck, Gustavo M; Bugarski, Branko; Leskošek-Čukalović, Ida; Vunduk, Jovana; Nikićević, Ninoslav; Nedović, Viktor

    2015-01-01

    The objectives of this study were to assess the differences in fermentative behaviour of two different strains of Saccharomyces cerevisiae (EC1118 and RC212) and to determine the differences in composition and sensory properties of raspberry wines fermented with immobilized and suspended yeast cells of both strains at 15 °C. Analyses of aroma compounds, glycerol, acetic acid and ethanol, as well as the kinetics of fermentation and a sensory evaluation of the wines, were performed. All fermentations with immobilized yeast cells had a shorter lag phase and faster utilization of sugars and ethanol production than those fermented with suspended cells. Slower fermentation kinetics were observed in all the samples that were fermented with strain RC212 (suspended and immobilized) than in samples fermented with strain EC1118. Significantly higher amounts of acetic acid were detected in all samples fermented with strain RC212 than in those fermented with strain EC1118 (0.282 and 0.602 g/l, respectively). Slightly higher amounts of glycerol were observed in samples fermented with strain EC1118 than in those fermented with strain RC212.

  4. Enhancement of chitosanase production by cell immobilization of Gongronella sp. JG

    Directory of Open Access Journals (Sweden)

    Pingping Zhang

    2013-01-01

    Full Text Available Chitosanase production of Gongronella sp. JG cells immobilized in calcium alginate gel and polyurethane foam was compared with that of the free cells, there was a 60% increase in the enzyme yield (2429 U/L compared to the highest yield obtained from free cells (1513 U/L. The optimal immobilization parameters (concentrations of sodium alginate, calcium chloride, bead inoculums, bead diameter, etc for the enhanced production of chitosanase were determined as: sodium alginate 2% (w/v, 0.1 M calcium chloride, inoculum 10 mL beads to 100 mL production media and 2.7 mm bead diameter. Maximum chitosanase production was achieved with initial pH of 5.5 and temperature of 30 ºC. The alginate beads had well stability, retained 85% ability of enzyme production even after 7 cycles of repeated batch fermentation. These results showed the immobilization technique was a feasible and economical method for chitosansase production by Gongronella sp. JG.

  5. The production of sorbitol by permeabilized and immobilized cells of Z. mobilis in sucrose

    Directory of Open Access Journals (Sweden)

    Josiane Alessandra Vignoli

    2006-07-01

    Full Text Available The production of sorbitol by permeabilized and immobilized cells of Zymomonas mobilis in Luffa cylindrica was investigated in sucrose medium. A full 2³ factorial design was used to verify the influence of each factor and its interactions. The cell permeabilization showed a significant and negative effect upon the production of sorbitol, while the time of cultivation and the immobilization process were significant and positive. The results demonstrated that the cell immobilization and the time of cultivation of 36 h presented higher production of sorbitol.Foi investigado a produção de sorbitol em meio de sacarose por células de Z. mobilis permeabilizadas e imobilizadas em Luffa cylindrica. Este trabalho avaliou o efeito da permeabilização de células de Z. mobilis tratadas com cetilmetilamoniobrometo e imobilizadas em Luffa cylindrinca. Um planejamento fatorial completo 2³ foi utilizado para verificar a influência dos fatores e suas interações. A permeabilização da célula mostrou um efeito significante e negativo sobre a produção de sorbitol, enquanto o tempo de cultivo e o processo de imobilização foram significantes e positivos. Os resultados mostraram que a imobilização das células e o tempo de cultivo de 36h forneceram concentração mais elevadas de sorbitol.

  6. Manipulation and Immobilization of a Single Fluorescence Nanosensor for Selective Injection into Cells

    Directory of Open Access Journals (Sweden)

    Hairulazwan Hashim

    2016-12-01

    Full Text Available Manipulation and injection of single nanosensors with high cell viability is an emerging field in cell analysis. We propose a new method using fluorescence nanosensors with a glass nanoprobe and optical control of the zeta potential. The nanosensor is fabricated by encapsulating a fluorescence polystyrene nanobead into a lipid layer with 1,3,3-trimethylindolino-6′-nitrobenzopyrylospiran (SP, which is a photochromic material. The nanobead contains iron oxide nanoparticles and a temperature-sensitive fluorescent dye, Rhodamine B. The zeta potential of the nanosensor switches between negative and positive by photo-isomerization of SP with ultraviolet irradiation. The positively-charged nanosensor easily adheres to a negatively-charged glass nanoprobe, is transported to a target cell, and then adheres to the negatively-charged cell membrane. The nanosensor is then injected into the cytoplasm by heating with a near-infrared (NIR laser. As a demonstration, a single 750 nm nanosensor was picked-up using a glass nanoprobe with optical control of the zeta potential. Then, the nanosensor was transported and immobilized onto a target cell membrane. Finally, it was injected into the cytoplasm using a NIR laser. The success rates of pick-up and cell immobilization of the nanosensor were 75% and 64%, respectively. Cell injection and cell survival rates were 80% and 100%, respectively.

  7. Bioethanol Production from Uncooked Raw Starch by Immobilized Surface-engineered Yeast Cells

    Science.gov (United States)

    Chen, Jyh-Ping; Wu, Kuo-Wei; Fukuda, Hideki

    Surface-engineered yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis α-amylase on the cell surface was used for direct production of ethanol from uncooked raw starch. By using 50 g/L cells during batch fermentation, ethanol concentration could reach 53 g/L in 7 days. During repeated batch fermentation, the production of ethanol could be maintained for seven consecutive cycles. For cells immobilized in loofa sponge, the concentration of ethanol could reach 42 g/L in 3 days in a circulating packed-bed bioreactor. However, the production of ethanol stopped thereafter because of limited contact between cells and starch. The bioreactor could be operated for repeated batch production of ethanol, but ethanol concentration dropped to 55% of its initial value after five cycles because of a decrease in cell mass and cell viability in the bioreactor. Adding cells to the bioreactor could partially restore ethanol production to 75% of its initial value.

  8. Cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Ghorbani, Farshid; Younesi, Habibollah; Esmaeili Sari, Abbas [Department of Environmental Science, Faculty of Natural Resources and Marine Sciences, Tarbiat Modares University, Noor, P.O. Box: 64414-356 (Iran); Najafpour, Ghasem [Department of Chemical Engineering, Faculty of Engineering, Noshirvani University of Technology, Babol (Iran)

    2011-02-15

    Sodium-alginate immobilized yeast was employed to produce ethanol continuously using cane molasses as a carbon source in an immobilized cell reactor (ICR). The immobilization of Saccharomyces cerevisiae was performed by entrapment of the cell cultured media harvested at exponential growth phase (16 h) with 3% sodium alginate. During the initial stage of operation, the ICR was loaded with fresh beads of mean diameter of 5.01 mm. The ethanol production was affected by the concentration of the cane molasses (50, 100 and 150 g/l), dilution rates (0.064, 0.096, 0.144 and 0.192 h{sup -1}) and hydraulic retention time (5.21, 6.94, 10.42 and 15.63 h) of the media. The pH of the feed medium was set at 4.5 and the fermentation was carried out at an ambient temperature. The maximum ethanol production, theoretical yield (Y{sub E/S}), volumetric ethanol productivity (Q{sub P}) and total sugar consumption was 19.15 g/l, 46.23%, 2.39 g l{sup -1} h{sup -1} and 96%, respectively. (author)

  9. Regulation of gene expression in Dictyostelium discoideum cells exposed to immobilized carbohydrates.

    Science.gov (United States)

    Bozzaro, S; Perlo, C; Ceccarelli, A; Mangiarotti, G

    1984-01-01

    When amoebae of Dictyostelium discoideum develop on gels of polyacrylamide that are derivatized with glucosides, they become capable of aggregation at the same time as cells not exposed to glucosides. However, the aggregation centers and streams of adherent cells formed on immobilized glucosides suddenly disintegrate. The cells repeatedly re-aggregate, but never form tight aggregates as they do on other substrata. Tight aggregates formed in the absence of glucosides disperse after their transfer to glucoside gels, and the cells undergo aggregation-disaggregation cycles. The formation of tight aggregates is correlated with the expression of specific post-aggregative poly(A) RNAs. These RNAs are not expressed in cells developing on glucoside gels, and the dispersal of tight aggregates on such gels is accompanied by the almost complete loss of these RNAs. A developmentally regulated membrane glycoprotein called contact site A, which is a marker of aggregation-competent cells, is normally expressed on glucoside gels. Cyclic AMP is also produced, indicating that the strong increase of adenylate cyclase activity during the preaggregation phase is not affected. In conclusion, cell contact with immobilized glucosides specifically inhibits postaggregative gene expression and arrests development at the aggregation stage.

  10. Highly sensitive determination of copper in HeLa cell using capillary electrophoresis combined with a simple cell extraction treatment.

    Science.gov (United States)

    Meng, Lingchen; Fang, Ziyuan; Lin, Jian; Li, Meixian; Zhu, Zhiwei

    2014-04-01

    A new separation system of capillary electrophoresis (CE1) for the highly sensitive determination of copper was established by using ethylenediaminetetraacetic acid (EDTA) as a complexing agent and employing cetyltrimethylammonium chloride (CTAC) as a capillary inner wall modifier. Benefitted from the combination of field-enhanced sample injection (FESI) method, a limit of detection (LOD) of 2.7 nM was obtained, which was much lower than that of the conventional methods. This made it possible to determine trace copper in HeLa cell only by a simple cell extraction (CE2) treatment. Two copper-extraction methods-acid-hydrolysis and freeze-thaw-were compared. Limited by the requirement of low ion strength in FESI, only the extract using freeze-thaw could be successfully applied in the determination. The effectiveness assessment of this CE(2)-FESI method was adopted by inductively coupled plasma-atomic emission spectrometry (ICP-AES) as a gold standard.

  11. Bioactivity of immobilized hyaluronic acid derivatives regarding protein adsorption and cell adhesion

    DEFF Research Database (Denmark)

    Köwitsch, Alexander; Yang, Yuan; Ma, Ning;

    2011-01-01

    Hyaluronic acid (HA) was chemically modified either by oxidation to obtain aldehyde-HA (aHA) or 3,3'-dithiobis(propanoic hydrazide) to obtain thiol-HA (tHA) that was covalently immobilized on model substrata such as amino-terminated surfaces or gold. Knowledge about the effect of modification...... affects cell growth and differentiation. A lower number and spreading of cells were observed on HA-modified surfaces compared to amino- and vinyl-terminated glass and silicon surfaces. Immunofluorescence microscopy also revealed that adhesion of fibroblast plated on HA-modified surfaces was mediated...

  12. Chitooligomer-Immobilized Biointerfaces with Micropatterned Geometries for Unidirectional Alignment of Myoblast Cells

    Directory of Open Access Journals (Sweden)

    Pornthida Poosala

    2016-01-01

    Full Text Available Skeletal muscle possesses a robust capacity to regenerate functional architectures with a unidirectional orientation. In this study, we successfully arranged skeletal myoblast (C2C12 cells along micropatterned gold strips on which chitohexaose was deposited via a vectorial chain immobilization approach. Hexa-N-acetyl-d-glucosamine (GlcNAc6 was site-selectively modified at its reducing end with thiosemicarbazide, then immobilized on a gold substrate in striped micropatterns via S–Au chemisorption. Gold micropatterns ranged from 100 to 1000 µm in width. Effects of patterning geometries on C2C12 cell alignment, morphology, and gene expression were investigated. Unidirectional alignment of C2C12 cells having GlcNAc6 receptors was clearly observed along the micropatterns. Decreasing striped pattern width increased cell attachment and proliferation, suggesting that the fixed GlcNAc6 and micropatterns impacted cell function. Possibly, interactions between nonreducing end groups of fixed GlcNAc6 and cell surface receptors initiated cellular alignment. Our technique for mimicking native tissue organization should advance applications in tissue engineering.

  13. The use of covalently immobilized stem cell factor to selectively affect hematopoietic stem cell activity within a gelatin hydrogel

    Science.gov (United States)

    Mahadik, B.P.; Haba, S. Pedron; Skertich, L.J.; Harley, B.A.C.

    2015-01-01

    Hematopoietic stem cells (HSCs) are a rare stem cell population found primarily in the bone marrow and responsible for the production of the body’s full complement of blood and immune cells. Used clinically to treat a range of hematopoietic disorders, there is a significant need to identify approaches to selectively expand their numbers ex vivo. Here we describe a methacrylamide-functionalized gelatin (GelMA) hydrogel for in vitro culture of primary murine HSCs. Stem cell factor (SCF) is a critical biomolecular component of native HSC niches in vivo and is used in large dosages in cell culture media for HSC expansion in vitro. We report a photochemistry based approach to covalently immobilize SCF within GelMA hydrogels via acrylate-functionalized polyethylene glycol (PEG) tethers. PEG-functionalized SCF retains the native bioactivity of SCF but can be stably incorporated and retained within the GelMA hydrogel over 7 days. Freshly-isolated murine HSCs cultured in GelMA hydrogels containing covalently-immobilized SCF showed reduced proliferation and improved selectivity for maintaining primitive HSCs. Comparatively, soluble SCF within the GelMA hydrogel network induced increased proliferation of differentiating hematopoietic cells. We used a microfluidic templating approach to create GelMA hydrogels containing gradients of immobilized SCF that locally direct HSC response. Together, we report a biomaterial platform to examine the effect of the local presentation of soluble vs. matrix-immobilized biomolecular signals on HSC expansion and lineage specification. This approach may be a critical component of a biomaterial-based artificial bone marrow to provide the correct sequence of niche signals to grow HSCs in the laboratory. PMID:26232879

  14. Alcohol production from whey permeate by immobilized and free cells of Kluyveromyces marxianus NCYC 179

    Energy Technology Data Exchange (ETDEWEB)

    Marwaha, S.S.; Kennedy, J.F.

    1984-04-01

    Comparative studies on the immobilized and free cells of Kluyveromyces marxianus NCYC 179 and Saccharomyces cerevisiae NCYC 240 were carried out to test their ability to metabolise whey lactose for ethanol production in batch fermentation. Complete utilisation of whey lactose (9.8%) resulting in 42.6 g ethanol/liter by K. marxianus NCYC 179 was observed, when fermentation was carried out under anaerobic conditions at pH 5.5, 30 degrees C temperature and 200 rev/minute. However, S. cerevisiae NCYC 240 proved to be an inefficient culture. Of the two gels, namely sodium alginate and K-carrageenan, monitored for their efficiency in entrapping the cells, sodium alginate at 2% (w/v) concentration was observed to retain a cell load of 120 g dry weight cell/liter effectively for optimum alcohol production. The immobilized K. marxianus NCYC 179 cells were examined in a packed bed reactor system under optimized fermentation conditions from batch fermenation experiments. Ethanol production efficiency was monitored at variable flow rates and the results are discussed. 11 references.

  15. Biochips for cell biology by combined dip-pen nanolithography and DNA-directed protein immobilization.

    Science.gov (United States)

    Arrabito, Giuseppe; Reisewitz, Stephanie; Dehmelt, Leif; Bastiaens, Philippe I; Pignataro, Bruno; Schroeder, Hendrik; Niemeyer, Christof M

    2013-12-20

    A general methodology for patterning of multiple protein ligands with lateral dimensions below those of single cells is described. It employs dip pen nanolithography (DPN) patterning of DNA oligonucleotides which are then used as capture strands for DNA-directed immobilization (DDI) of oligonucleotide-tagged proteins. This study reports the development and optimization of PEG-based liquid ink, used as carrier for the immobilization of alkylamino-labeled DNA oligomers on chemically activated glass surfaces. The resulting DNA arrays have typical spot sizes of 4-5 μm with a pitch of 12 μm micrometer. It is demonstrated that the arrays can be further functionalized with covalent DNA-streptavidin (DNA-STV) conjugates bearing ligands recognized by cells. To this end, biotinylated epidermal growth factor (EGF) is coupled to the DNA-STV conjugates, the resulting constructs are hybridized with the DNA arrays and the resulting surfaces used for the culturing of MCF-7 (human breast adenocarcinoma) cells. Owing to the lateral diffusion of transmembrane proteins in the cell's plasma membrane, specific recruitment and concentration of EGF receptor can be induced specifically at the sites where the ligands are bound on the solid substrate. This is a clear demonstration that this method is suitable for precise functional manipulations of subcellular areas within living cells.

  16. A chip-type thin-layer electrochemical cell coupled with capillary electrophoresis for online separation of electrode reaction products

    Energy Technology Data Exchange (ETDEWEB)

    He, Jian-Bo, E-mail: jbhe@hfut.edu.cn; Cui, Ting; Zhang, Wen-Wen; Deng, Ning

    2013-07-05

    Graphical abstract: -- Highlights: •A new coupling of thin-layer electrolysis with capillary electrophoresis (CE). •Rapid electrolysis, direct sampling followed by online CE separation. •At least 13 products of quercetin oxidation were separated. •Thermodynamic and kinetic parameters were determined from CE peak areas. -- Abstract: A coupling technique of thin-layer electrolysis with high-performance capillary electrophoresis/UV–vis technique(EC/HPCE/UV–vis) is developed for online separation and determination of electrode reaction products. A chip-type thin-layer electrolytic (CTE) cell was designed and fabricated, which contains a capillary channel and a background electrolyte reservoir, allowing rapid electrolysis, direct sampling and online electrophoretic separation. This chip-type setup was characterized based on an electrophoresis expression of Nernst equation that was applied to the redox equilibrium of o-tolidine at different potentials. The utility of the method was demonstrated by separating and determining the electro-oxidation products of quercetin in different pH media. Two main products were always found in the studied time, potential and pH ranges. The variety of products increased not only with increasing potential but also with increasing pH value, and in total, at least 13 products were observed in the electropherograms. This work illustrates a novel example of capillary electrophoresis used online with thin-layer electrolysis to separate and detect electrode reaction products.

  17. Nitrilase-catalyzed conversion of (R,S)-mandelonitrile by immobilized recombinant Escherichia coli cells harboring nitrilase.

    Science.gov (United States)

    Zhang, Xin-Hong; Liu, Zhi-Qiang; Xue, Ya-Ping; Xu, Ming; Zheng, Yu-Guo

    2016-07-01

    (R)-(-)-Mandelic acid (R-MA) is widely used both as a versatile intermediate for pharmaceuticals and a resolving agent in chiral resolution processes. In the current study, to improve the stability of operation, recombinant Escherichia coli cells expressing nitrilase from Alcaligenes faecalis were immobilized for the enantioselective hydrolysis of (R,S)-mandelonitrile to R-MA. Different immobilization methods including entrapment matrices, entrapment matrices cross-linked by cross-linking and polymerization agents, and direct cross-linking cells using glutaraldehyde (GA) or bionic silicon were investigated. To facilitate industrial solid-liquid separation, the direct cross-linking recombinant E. coli cells using diatomite/GA/polyethyleneimine with 135.95% relative activity compared with free cells was chosen using water as the reaction medium. The operational stability of the immobilized cells was obviously superior to that of free cells, without significant activity loss after 28 cycles of batch reaction and the successive production of R-MA could reach 1.88 M. Moreover, the immobilized cells showed good storage stability with about 52% relative activity after storing for 30 days at 4 °C. Therefore, the immobilized biocatalyst is very promising for upscale production of optically pure R-MA with high performance and low cost.

  18. Chitin hydrolysis assisted by cell wall degrading enzymes immobilized of Thichoderma asperellum on totally cinnamoylated D-sorbitol beads.

    Science.gov (United States)

    Fernandes, Kátia F; Cortijo-Triviño, David; Batista, Karla A; Ulhoa, Cirano J; García-Ruiz, Pedro A

    2013-07-01

    In this study, cell wall degrading enzymes produced by Thrichoderma asperellum (TCWDE) were immobilized on totally cinnamoylated D-sorbitol (TCNSO) beads and used for chitin hydrolysis. In order to optimize immobilization efficiency, the reaction time was varied from 2 to 12 h and reactions were conducted in the presence or absence of Na2SO4. Immobilized enzymes were analysed concerning to thermal and operational stability. Immobilization in presence of Na2SO4 was 54% more efficient than immobilization in absence of salt. After optimization, 32% of the total enzyme offered was immobilized, with 100% of bounding efficiency, measured as the relation between protein and enzyme immobilized. Free and TCNSO-TCWDE presented very similar kinetics with maximum hydrolysis reached at 90 min of reaction. Thermal stability of both free and TCNSO-TCWDE was similar, with losses in activity after 55 °C. Moreover, free and TCNSO-TCWDE retained 100% activity after 3h incubation at 55 °C. TCNSO-TCWDE were used in a bath-wise reactor during 14 cycles, producing 1825 μg of N-acetylglucosamine (NAG) maintaining 83% of initial activity.

  19. Secretomic analysis of large cell lung cancer cell lines using two-dimensional gel electrophoresis coupled to mass spectrometry

    Directory of Open Access Journals (Sweden)

    Zahra Yousefi

    2012-10-01

    Full Text Available

    The secretome of cancer cells is a valuable source of biomarkers that can ultimately reach the serum or other body fluids. Cancer biomarkers can facilitate early diagnosis and monitoring of the disease, contribute to our understanding of tumor biology, and support the development of more efficient therapies. Recently, high-throughput proteomic analysis of the conditioned media of cancer cell lines has shown potential to identify novel biomarkers in cancer. We used two-dimensional gel electrophoresis coupled to liquid chromatography tandem mass spectrometry to identify the secretome of the large cell lung cancer cell lines QU-DB and Mehr-80, which they were established from a Canadian and a Persian patient, respectively. A total of 130 distinct protein species were identified. Of these, 124 were previously found in serum or other body fluids, the membrane compartment or conditioned media of other cancer cell lines. Some of the proteins that we identified, e.g. IL-6, triosephosphate isomerase, PGP9.5, α-enolase, Dickkopf-1, and peroxiredoxin-1 are known putative serum markers for lung cancer, whereas others might be candidate markers for further validation in lung cancer body fluids such as IL-25, stathmin, vimentin, peptidyl-prolyl cis-trans isomerase A, transgelin-2, and chloride intracellular channel protein 4.

  20. An Evaluation of Kinetic Parameters of Cadmium and Copper Biosorption by Immobilized Cells

    Directory of Open Access Journals (Sweden)

    Nelly Georgieva

    2007-10-01

    Full Text Available Bioremediation is the use of living organisms to reduce or eliminate environmental hazards resulting from the accumulation of toxic chemicals and other hazardous wastes. This technology is based on the utilization of microorganisms to transform organic and inorganic compounds. The filamentous yeast Trichosporon cutaneum strain R57, immobilized and free cells was cultivated as batch culture on a liquid medium in the presence of various concentrations of cadmium and copper ions. The simultaneous uptake and accumulation of Cd2+ and Cu2+ ions by Tr. cutaneum cells depending on the initial concentration of Cd2+ and Cu2+ in the medium were studied. The potential use of the free and immobilized cells of Trichosporon cutaneum to remove cadmium and copper ions, from aqueous solutions was evaluated. Two important physicochemical aspects for the evaluation of the sorption process as a unit operation are the equilibrium of sorption and the kinetics. The Cd2+ and Cu2+ ions biosorption capacities of all tested adsorbent were presented as a function of the initial concentration of metal ions within the aqueous biosorption medium. The individual, as well as bicomponent sorption kinetics of copper and cadmium ions by immobilised cells of Trichosporon cutaneum R57 is presented. A second order kinetic model obtains kinetic parameters for the copper and cadmium ions.

  1. Phytoremediation of Benzophenone and Bisphenol A by Glycosylation with Immobilized Plant Cells

    Directory of Open Access Journals (Sweden)

    Kei Shimoda

    2009-01-01

    Full Text Available Benzophenone and bisphenol A are environmental pollutions, which have been listed among “chemicals suspected of having endocrine disrupting effects” by the World Wildlife Fund, the National Institute of Environmental Health Sciences in the USA and the Japanese Environment Agency. The cultured cells of Nicotiana tabacum glycosylated benzophenone to three glycosides, 4-O-β-D-glucopyranosylbenzophenone (9%, diphenylmethyl β-D-glucopyranoside (14%, and diphenylmethyl 6-O-(β-D-glucopyranosyl-β-D-glucopyranoside (12% after 48 h incubation. On the other hand, incubation of benzophenone with immobilized cells of N. tabacum in sodium alginate gel gave products in higher yields, i.e. the yields of 4-O-β-D-glucopyranosylbenzophenone, diphenylmethyl β-D-glucopyranoside, and diphenylmethyl 6-O-(β-D-glucopyranosyl-β-D-glucopyranoside were 15, 27, and 22%, respectively. Bisphenol A was converted into three glycosides, 2,2-bis(4-β-D-glucopyranosyloxyphenylpropane (16%, 2-(4-β-D-glucopyranosyloxy-3-hydroxyphenyl-2-(4-β-D-gluco- pyranosyloxyphenyl propane (8%, and 2-(3-β-D-glucopyranosyloxy-4-hydroxyphenyl-2-(4-β-D-glucopyranosyloxyphe nylpropane (5%. Also the use of immobilized N. tabacum cells improved the yield of products; the glycosylation of bisphenol A with immobilized N. tabacum gave 2,2-bis(4-β-D-glucopyranosyloxyphenylpropane (24%, 2-(4-β-D-gluco- pyranosyloxy-3-hydroxyphenyl-2-(4-β-D-glucopyranosyloxyphenyl propane (15%, and 2-(3-β-D-glucopyranosyloxy- 4-hydroxyphenyl-2-(4-β-D-glucopyranosyloxyphenylpropane (11%.

  2. Simultaneous Alcoholic and Malolactic Fermentations by Saccharomyces cerevisiae and Oenococcus oeni Cells Co-immobilized in Alginate Beads

    Science.gov (United States)

    Bleve, Gianluca; Tufariello, Maria; Vetrano, Cosimo; Mita, Giovanni; Grieco, Francesco

    2016-01-01

    Malolactic fermentation (MLF) usually takes place after the end of alcoholic fermentation (AF). However, the inoculation of lactic acid bacteria together with yeast starter cultures is a promising system to enhance the quality and safety of wine. In recent years, the use of immobilized cell systems has been investigated, with interesting results, for the production of different fermented foods and beverages. In this study we have carried out the simultaneous immobilization of Saccharomyces cerevisiae and Oenococcus oeni in alginate beads and used them in microvinifications tests to produce Negroamaro wine. The process was monitored by chemical and sensorial analyses and dominance of starters and cell leaking from beads were also checked. Co-immobilization of S. cerevisiae and O. oeni allowed to perform an efficient fermentation process, producing low volatile acidity levels and ethanol and glycerol concentrations comparable with those obtained by cell sequential inoculum and co-inoculum of yeast and bacteria cells in free form. More importantly, co-immobilization strategy produced a significant decrease of the time requested to complete AF and MLF. The immobilized cells could be efficiently reused for the wine fermentation at least three times without any apparent loss of cell metabolic activities. This integrated biocatalytic system is able to perform simultaneously AF and MLF, producing wines similar in organoleptic traits in comparison with wines fermented following traditional sequential AF and MLF with free cell starters. The immobilized-cell system, that we here describe for the first time in our knowledge, offers many advantages over conventional free cell fermentations, including: (i) elimination of non-productive cell growth phases; (ii) feasibility of continuous processing; (iii) re-use of the biocatalyst. PMID:27379072

  3. Study on Immobilized Algal Cells for Treatment and Recycle of Refinery Wastewater

    Institute of Scientific and Technical Information of China (English)

    Yu Baocheng; Liu Deqi; Dong Lihua; Li Gang

    2005-01-01

    Compared to the algal oxidation pond, treatment of wastewater using the immobilized algal cell technology has excellent effect, which not only can effectively avoid the disadvantage of oxidation pond,but can also remarkably improve the efficiency of treating system and the effluent quality. When the treating system operates under an optimal control conditions, such as a 55% loading rate, an illumination intensity of 2500-3500 lux and a hydraulic residence time of 4 hours, the COD and ammonia nitrogen removal can reach 90%. Water after deep treatment can comply with the requirement of the refinery for the quality of recycled water.

  4. Acetone-butanol-ethanol (ABE) fermentation in an immobilized cell trickle bed reactor.

    Science.gov (United States)

    Park, C H; Okos, M R; Wankat, P C

    1989-06-05

    Acetone-butanol-ethanol (ABE) fermentation was successfully carried out in an immobilized cell trickle bed reactor. The reactor was composed of two serial columns packed with Clostridium acetobutylicum ATCC 824 entrapped on the surface of natural sponge segments at a cell loading in the range of 2.03-5.56 g dry cells/g sponge. The average cell loading was 3.58 g dry cells/g sponge. Batch experiments indicated that a critical pH above 4.2 is necessary for the initiation of cell growth. One of the media used during continuous experiments consisted of a salt mixture alone and the other a nutrient medium containing a salt mixture with yeast extract and peptone. Effluent pH was controlled by supplying various fractions of the two different types of media. A nutrient medium fraction above 0.6 was crucial for successful fermentation in a trickle bed reactor. The nutrient medium fraction is the ratio of the volume of the nutrient medium to the total volume of nutrient plus salt medium. Supplying nutrient medium to both columns continuously was an effective way to meet both pH and nutrient requirement. A 257-mL reactor could ferment 45 g/L glucose from an initial concentration of 60 g/L glucose at a rate of 70 mL/h. Butanol, acetone, and ethanol concentrations were 8.82, 5.22, and 1.45 g/L, respectively, with a butanol and total solvent yield of 19.4 and 34.1 wt %. Solvent productivity in an immobilized cell trickle bed reactor was 4.2 g/L h, which was 10 times higher than that obtained in a batch fermentation using free cells and 2.76 times higher than that of an immobilized CSTR. If the nutrient medium fraction was below 0.6 and the pH was below 4.2, the system degenerated. Oxygen also contributed to the system degeneration. Upon degeneration, glucose consumption and solvent yield decreased to 30.9 g/L and 23.0 wt %, respectively. The yield of total liquid product (40.0 wt %) and butanol selectivity (60.0 wt %) remained almost constant. Once the cells were degenerated

  5. Oxygen supply for CHO cells immobilized on a packed-bed of Fibra-Cel disks.

    Science.gov (United States)

    Meuwly, F; Loviat, F; Ruffieux, P-A; Bernard, A R; Kadouri, A; von Stockar, U

    2006-03-05

    Packed-bed bioreactors (PBR) have proven to be efficient systems to culture mammalian cells at very high cell density in perfusion mode, thus leading to very high volumetric productivity. However, the immobilized cells must be continuously supplied with all nutrients in sufficient quantities to remain viable and productive over the full duration of the perfusion culture. Among all nutrients, oxygen is the most critical since it is present at very low concentration due to its low solubility in cell culture medium. This work presents the development of a model for oxygenation in a packed-bed bioreactor system. The experimental system used to develop the model was a packed-bed of Fibra-Cel disk carriers used to cultivate Chinese Hamster Ovary cells at high density ( approximately 6.1 x 10(7) cell/mL) in perfusion mode. With the help of this model, it was possible to identify if a PBR system is operated in optimal or sub-optimal conditions. Using the model, two options were proposed, which could improve the performance of the basal system by about twofold, that is, by increasing the density of immobilized cells per carrier volume from 6.1 x 10(7) to 1.2 x 10(8) cell/mL, or by increasing the packed-bed height from 0.2 to 0.4 m. Both strategies would be rather simple to test and implement in the packed-bed bioreactor system used for this study. As a result, it would be possible to achieve a substantial improvement of about twofold higher productivity as compared with the basal conditions.

  6. Enhancing anticoagulation and endothelial cell proliferation of titanium surface by sequential immobilization of poly(ethylene glycol) and collagen

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Chang-Jiang, E-mail: swjtupcj@163.com; Hou, Yan-Hua; Ding, Hong-Yan; Dong, Yun-Xiao

    2013-12-15

    In the present study, poly(ethylene glycol) (PEG) and collagen I were sequentially immobilized on the titanium surface to simultaneously improve the anticoagulation and endothelial cell proliferation. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy analysis confirmed that PEG and collagen I were successfully immobilized on the titanium surface. Water contact angle results suggested the excellent hydrophilic surface after the immobilization. The anticoagulation experiments demonstrated that the immobilized PEG and collagen I on the titanium surface could not only obviously prevent platelet adhesion and aggregation but also prolong activated partial thromboplastin time (APTT), leading to the improved blood compatibility. Furthermore, immobilization of collagen to the end of PEG chain did not abate the anticoagulation. As compared to those on the pristine and PEG-modified titanium surfaces, endothelial cells exhibited improved proliferative profiles on the surface modified by the sequential immobilization of PEG and collagen in terms of CCK-8 assay, implying that the modified titanium may promote endothelialization without abating the blood compatibility. Our method may be used to modify the surface of blood-contacting biomaterials such as titanium to promote endothelialization and improve the anticoagulation, it may be helpful for development of the biomedical devices such as coronary stents, where endothelializaton and excellent anticoagulation are required.

  7. Enhancing anticoagulation and endothelial cell proliferation of titanium surface by sequential immobilization of poly(ethylene glycol) and collagen

    Science.gov (United States)

    Pan, Chang-Jiang; Hou, Yan-Hua; Ding, Hong-Yan; Dong, Yun-Xiao

    2013-12-01

    In the present study, poly(ethylene glycol) (PEG) and collagen I were sequentially immobilized on the titanium surface to simultaneously improve the anticoagulation and endothelial cell proliferation. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy analysis confirmed that PEG and collagen I were successfully immobilized on the titanium surface. Water contact angle results suggested the excellent hydrophilic surface after the immobilization. The anticoagulation experiments demonstrated that the immobilized PEG and collagen I on the titanium surface could not only obviously prevent platelet adhesion and aggregation but also prolong activated partial thromboplastin time (APTT), leading to the improved blood compatibility. Furthermore, immobilization of collagen to the end of PEG chain did not abate the anticoagulation. As compared to those on the pristine and PEG-modified titanium surfaces, endothelial cells exhibited improved proliferative profiles on the surface modified by the sequential immobilization of PEG and collagen in terms of CCK-8 assay, implying that the modified titanium may promote endothelialization without abating the blood compatibility. Our method may be used to modify the surface of blood-contacting biomaterials such as titanium to promote endothelialization and improve the anticoagulation, it may be helpful for development of the biomedical devices such as coronary stents, where endothelializaton and excellent anticoagulation are required.

  8. Characteristics of an immobilized yeast cell system using very high gravity for the fermentation of ethanol.

    Science.gov (United States)

    Ji, Hairui; Yu, Jianliang; Zhang, Xu; Tan, Tianwei

    2012-09-01

    The characteristics of ethanol production by immobilized yeast cells were investigated for both repeated batch fermentation and continuous fermentation. With an initial sugar concentration of 280 g/L during the repeated batch fermentation, more than 98% of total sugar was consumed in 65 h with an average ethanol concentration and ethanol yield of 130.12 g/L and 0.477 g ethanol/g consumed sugar, respectively. The immobilized yeast cell system was reliable for at least 10 batches and for a period of 28 days without accompanying the regeneration of Saccharomyces cerevisiae inside the carriers. The multistage continuous fermentation was carried out in a five-stage column bioreactor with a total working volume of 3.75 L. The bioreactor was operated for 26 days at a dilution rate of 0.015 h(-1). The ethanol concentration of the effluent reached 130.77 g/L ethanol while an average 8.18 g/L residual sugar remained. Due to the high osmotic pressure and toxic ethanol, considerable yeast cells died without regeneration, especially in the last two stages, which led to the breakdown of the whole system of multistage continuous fermentation.

  9. Repeated batch cell-immobilized system for the biotechnological production of xylitol as a renewable green sweetener.

    Science.gov (United States)

    Sarrouh, Boutros; da Silva, Silvio Silvério

    2013-04-01

    The present paper studies the biotechnological production of xylitol using sugarcane bagasse hydrolysate in a repeated batch fermentation system with immobilized cells of Candida guilliermondii FTI20037. Immobilized cell system is considered as an attractive alternative to reuse the well-grown and adapted yeast cells in a new fresh fermentation media, without the need of the inoculum stage. In this work, seven repeated batches were performed in a fluidized bed bioreactor using immobilized cells in calcium alginate beads. According to the obtained results it was observed that the immobilized cells of C. guilliermondii can be reused for six successive batches maintaining an average xylitol yield (Y(p/s)) of 0.7 g/L and a volumetric productivity (Q(p)) of 0.42 g/Lh at the end of 432 h of fermentation. On the other hand, in the seventh batch (504 h), a decrease of 44 % in the final concentration of xylitol was observed. This reduction can be explained by the possible diffusion and accumulation of insoluble substances, found in the hemicellulosic hydrolysate, in the interior of the immobilization support resulting in substrate mass transfer limitations.

  10. Immobilized Kluyveromyces marxianus cells in carboxymethyl cellulose for production of ethanol from cheese whey: experimental and kinetic studies.

    Science.gov (United States)

    Roohina, Fatemeh; Mohammadi, Maedeh; Najafpour, Ghasem D

    2016-09-01

    Cheese whey fermentation to ethanol using immobilized Kluyveromyces marxianus cells was investigated in batch and continuous operation. In batch fermentation, the yeast cells were immobilized in carboxymethyl cellulose (CMC) polymer and also synthesized graft copolymer of CMC with N-vinyl-2-pyrrolidone, denoted as CMC-g-PVP, and the efficiency of the two developed cell entrapped beads for lactose fermentation to ethanol was examined. The yeast cells immobilized in CMC-g-PVP performed slightly better than CMC with ethanol production yields of 0.52 and 0.49 g ethanol/g lactose, respectively. The effect of supplementation of cheese whey with lactose (42, 70, 100 and 150 g/l) on fermentative performance of K. marxianus immobilized in CMC beads was considered and the results were used for kinetic studies. The first order reaction model was suitable to describe the kinetics of substrate utilization and modified Gompertz model was quite successful to predict the ethanol production. For continuous ethanol fermentation, a packed-bed immobilized cell reactor (ICR) was operated at several hydraulic retention times; HRTs of 11, 15 and 30 h. At the HRT of 30 h, the ethanol production yield using CMC beads was 0.49 g/g which implies that 91.07 % of the theoretical yield was achieved.

  11. Investigating metal removal potential by Effective Microorganisms (EM) in alginate-immobilized and free-cell forms.

    Science.gov (United States)

    Ting, Adeline Su Yien; Rahman, Nurul Hidayah Abdul; Isa, Mohamed Ikmal Hafiz Mahamad; Tan, Wei Shang

    2013-11-01

    Metal removal potential of both alginate-immobilized and free-cells of Effective Microorganisms (EM-1™ Inoculant) was investigated in this study. Results revealed that removal of Cr(III), Cu(II) and Pb(II) followed a similar trend where alginate-immobilized EM were more efficient compared to free-cells of EM. For these metals, 0.940, 2.695 and 4.011 mg g(-1) of Cr(III), Cu(II) and Pb(II) were removed compared to only 0.160, 0.859 and 0.755 mg ml(-1) removed by free-cells, respectively. The higher efficiency of alginate-immobilized EM was primarily attributed to the alginate matrix. This was evident when both alginate-immobilized EM and plain alginate beads (without EM), were not significantly different in their removal efficacies. Presence of alginate also enhanced the use of the biosorbents as maximum metal sorption was achieved after 120 min as opposed to only 60 min for free-cells. EM per se in immobilized or free-cell forms did not enhance metal removal efficacy.

  12. Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

    Energy Technology Data Exchange (ETDEWEB)

    Gikas, P.; Livingston, A.G. (Imperial Coll., London (United Kingdom). Dept. of Chemical Engineering)

    1993-12-01

    This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bio-reactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h[sup [minus]1] in the CST bioreactor and between 0.111 and 0.500h[sup [minus]1] in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g[sup [minus]1] dry weight (dw) as dilution rate increases from 0.027 to 0.115 h[sup [minus]1]. At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g[sup [minus]1] dw, which is assumed to be the quantity of ATP in 100% viable biomass, In the TPAL bioreactor, the ATP level increased with dilution rat in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g[sup [minus]1] dw at dilution rates between 0.111 and 0.200 h[sup [minus]1] to approximately 0.119 mg ATP g[sup [minus]1] dw at dilution rates between 0.300 and 0.500 h[sup [minus]1].

  13. Application of immobilized cell preparation obtained from biomass of Gluconacetobacter xylinus bacteria in biotransformation of glycerol to dihydroxyacetone

    Directory of Open Access Journals (Sweden)

    Lidia Stasiak-Różańska

    2011-03-01

    Full Text Available Introduction. Dihydroxyacetone (DHA, being a product of glycerol oxidation by acetic acid bacteria, is an important compound widely applied in the cosmetic, food, and pharmaceutical industry, as well as in medicine. Biotransformation of glycerol to DHA is catalyzed by glycerol dehydrogenase (GlyDH, EC 1.1.1.6 bound with the cytoplasmic membrane of bacteria. An attempt was undertaken in this study to conduct glycerol biotransformation with immobilized fractions of a cell preparation with GlyDH activity. The content of dihydroxyacetone obtained with the cell preparation was compared with its content achieved in the reaction with immobilized viable cells of G. xylinus. Material and methods. Cell walls of Gluconacetobacter xylinus bacteria were disintegrated enzymatically. The resultant preparation was immobilized on calcium alginate or first separated into two fractions (precipitate and supernatant by centrifugation and then immobilized. DHA content was determined colorimetrically after the reaction with 3,5-dinitrosalicilic acid. Glycerol content was assayed with the refractometric method. Results. After 20 days of the process, the concentration of DHA obtained with immobilized whole cells reached 25 g/l. In turn, the content of DHA obtained in the same period with immobilized fractions of the cell preparation accounted for 16.9 g/l and 8.95 g/l (depending on the fraction applied. Conclusions. DHA may be obtained in the process independent of G. xylinus metabolic activity using a preparation which displays the catalytic activity of glycerol dehydrogenase and obtained as a result of disintegration of live bacterial cells. The application of such a preparation may in the future eliminate technological problems posed by the presence of bacterial cells and their metabolites in the culture medium.

  14. The application of single cell gel electrophoresis or comet assay to human monitoring studies Aplicacion de la electroforesis unicelular o ensayo cometa en estudios de monitoreo humano

    OpenAIRE

    1999-01-01

    Objective. In the search of new human genotoxic biomarkers, the single cell gel electrophoresis assay has been proposed as a sensible alternative. Material and methods. This technique detects principally single strand breaks as well as alkali-labile and repair-retarded sites. Results. Herein we present our experience using the single cell gel electrophoresis assay in human population studies, both occupationally and environmentally exposed. Conclusions. We discuss the assay feasibility as a g...

  15. DNA damaging effects of carbofuran and its main metabolites on mice by micronucleus test and single cell gel electrophoresis

    Institute of Scientific and Technical Information of China (English)

    ZHOU Pei; LIU Baofeng; LU Yitong

    2005-01-01

    The DNA damaging effects of the carbamate pesticide carbofuran and its four metabolites (carbofuranphenol, 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran) on mice were evaluated by single cell gel electrophoresis (SCGE) assay and micronucleus test. KM mice were exposed to test compounds with different doses of 0.1, 0.2 and 0.4mg/kg through intraperitoneal injection two times with an internal of 24 h, and then killed by cervical dislocation 6 h after the second injection. In SCGE assay, isolated mice peripheral blood lymphocytes were employed to determine DNA damaging degree after a 1 h treatment by test compounds and a following electrophoresis. Carbofuran and carbofuranphenol showed negative results in both test and had no obvious toxicity. 3-hydrocarbofuran and nitrosocarbofuran were positive.3-ketocarbofuran could not induce micronucleus formation but caused significant DNA migration in SCGE test. These tests revealed that 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran are potential mutagesis and further research is needed.

  16. Thienamycin production by immobilized cells of Streptomyces cattleya in a bubble column.

    Science.gov (United States)

    Arcuri, E J; Nichols, J R; Brix, T S; Santamarina, V G; Buckland, B C; Drew, S W

    1983-10-01

    A novel 2.0-L columnar reactor has been developed for the production of thienamycin by cells of Streptomyces cattleya attached to celite particles. Successful immobilization of cells was achieved by operating the column continuously at a high dilution rate during the growth phase. Scanning electron micrographs of the celite particles indicate the involvement of subcellular fibrils in the attachment of cells to the solid surfaces. Reactor operation was divided into two distinct phases-a growth phase and a production phase. The kinetics of attached growth and thienamycin production were found to be strongly influenced by nutrient concentrations. The influences of nutrient concentration on CO(2) production and thienamycin production during both the growth phase and the production phase are discussed.

  17. Two-dimensional gel electrophoresis for controlling and comparing culture supernatants of mammalian cell culture productions systems.

    Science.gov (United States)

    Wimmer, K; Harant, H; Reiter, M; Blüml, G; Gaida, T; Katinger, H

    1994-01-01

    A recombinant Chinese hamster ovary cell line, producing human erythropoietin, was cultivated in a continuous mode in a stirred tank reactor applying different dilution rates. In order to monitor the stability of this expression system, product and non-product proteins of the cell culture supernatant were analyzed by two-dimensional electrophoresis. The consistency of the isoforms of the recombinant product was determined by western blot combined with specific staining. The same cell line was propagated in a high cell density cultivation system based on macro-cell-aggregates. The patterns of secreted proteins of the cell line cultivated in the different systems were compared in order to detect modifications in protein expression of the product and of non product proteins relevant for cell culture supernatant. Hardly any alterations in two-dimensional pattern were detectable. The isoforms of erythropoietin, as well as the overall pattern of secreted proteins, detectable with the two-dimensional electrophoresis method were remarkably stable under different cultivation conditions.

  18. Fermentation efficiency of cells immobilized on delignified brewers' spent grains after low- and high-temperature thin layer thermal drying.

    Science.gov (United States)

    Tsaousi, Konstantina; Koutinas, Athanasios A; Bekatorou, Argyro; Loukatos, Paul

    2010-09-01

    Low-cost dried yeasts immobilized on delignified brewers' spent grains for use in wine making and brewing were produced by simple thermal drying techniques. To optimize the thermal drying process, vacuum and air stream conditions were examined. Drying of thin layers of the biocatalysts was performed at low (30-38 degrees C) and high temperatures (40-70 degrees C). The fermentation efficiency of the thermally dried biocatalysts was acceptable, with immobilized cells showing a significantly higher thermotolerance compared with free cells. Immobilized cells dried at high temperatures presented slightly improved glucose fermentation efficiency compared with the low-temperature dried biocatalysts. Gas chromatography-mass spectrometry analysis of aroma volatiles of the fermented products revealed an increase of esters, lower higher alcohol formation, and significantly lower concentration of carbonylic compounds.

  19. Identification of two-dimensional electrophoresis-separated proteins in human hepatoma cell by electrospray ion trap mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    As one of the most important analytical methods in proteome research, mass spectrometry was utilized to identify proteins separated by two-dimensional electrophoresis in the human hepatoma cell line BEL-7404. The protein spots were excised from the gel, followed by in-gel digestion, and the peptide mappings were analyzed by liquid chromatography electrospray ion trap mass spectrometer. Nine proteins were identified via database searching, according to the molecular weights and amino acid sequences of peptides, among which two proteins have not been identified in the other liver-cell database. The sequence coverage was 21%-72%. Furthermore, the relationship between the expressed proteins and the liver carcinoma was discussed.

  20. Bioethanol production from mixed sugars by Scheffersomyces stipitis free and immobilized cells, and co-cultures with Saccharomyces cerevisiae.

    Science.gov (United States)

    De Bari, Isabella; De Canio, Paola; Cuna, Daniela; Liuzzi, Federico; Capece, Angela; Romano, Patrizia

    2013-09-25

    Bioethanol can be produced from several biomasses including lignocellulosic materials. Besides 6-carbon sugars that represent the prevalent carbohydrates, some of these feedstocks contain significant amounts of 5-carbon sugars. One common limit of the major part of the xylose-fermenting yeasts is the diauxic shift between the uptake of glucose and xylose during the fermentation of mixed syrups. Thus, optimized fermentation strategies are required. In this paper the ability of Scheffersomyces stipitis strain NRRLY-11544 to ferment mixed syrups with a total sugar concentration in the range 40-80 g/L was investigated by using mono cultures, co-cultures with Saccharomyces cerevisiae strain Bakers Yeast Type II and single cultures immobilized in silica-hydrogel films. The experimental design for the fermentations with immobilized cells included the process analysis in function of two parameters: the fraction of the gel in the broth and the concentration of the cells loaded in the gel. Furthermore, for each total sugars level, the fermentative course of S. stipitis was analyzed at several glucose-to xylose ratios. The results indicated that the use of S. stipitis and S. cerevisiae in free co-cultures ensured faster processes than single cultures of S. stipitis either free or immobilized. However, the rapid production of ethanol by S. cerevisiae inhibited S. stipitis and caused a stuck of the process. Immobilization of S. stipitis in silica-hydrogel increased the relative consumption rate of xylose-to-glucose by 2-6 times depending on the composition of the fermentation medium. Furthermore the films performances appeared stable over three weeks of continuous operations. However, on the whole, the final process yields obtained with the immobilized cells were not meaningfully different from that of the free cells. This was probably due to concurrent fermentations operated by the cells released in the broth. Optimization of the carrier characteristics could improve the

  1. Assessing CMT cell line stability by two dimensional polyacrylamide gel electrophoresis and mass spectrometry based proteome analysis

    DEFF Research Database (Denmark)

    Zhang, Kelan; Wrzesinski, Krzysztof; Fey, Stephen J;

    2008-01-01

    Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by mass spectrometric identification of the proteins in the protein spots has become a central tool in proteomics. CMT167(H), CMT64(M) and CMT170(L) cell lines, selected from a spontaneous mouse lung adenocarcinoma, with high......-, middle- or low-metastatic potential have been characterized in vivo. In this study, the comprehensive protein expression profiles of the CMT cell lines were analyzed at passages 5, 15 and 35 in order to assess the cell line stability. During the passages 5 to 15, the expression profiles of CMT cells...... to be a useful tool for assessing differences in cell line stability. This approach provided a tool to select the best cell line and optimal subculture period for studies of cancer related phenomena and for testing the effect of potential anticancer drugs....

  2. A Hydrogel Bridge Incorporating Immobilized Growth Factors and Neural Stem/Progenitor Cells to Treat Spinal Cord Injury.

    Science.gov (United States)

    Li, Hang; Ham, Trevor R; Neill, Nicholas; Farrag, Mahmoud; Mohrman, Ashley E; Koenig, Andrew M; Leipzig, Nic D

    2016-04-06

    Spinal cord injury (SCI) causes permanent, often complete disruption of central nervous system (CNS) function below the damaged region, leaving patients without the ability to regenerate lost tissue. To engineer new CNS tissue, a unique spinal cord bridge is created to deliver stem cells and guide their organization and development with site-specifically immobilized growth factors. In this study, this bridge is tested, consisting of adult neural stem/progenitor cells contained within a methacrylamide chitosan (MAC) hydrogel and protected by a chitosan conduit. Interferon-γ (IFN-γ) and platelet-derived growth factor-AA (PDGF-AA) are recombinantly produced and tagged with an N-terminal biotin. They are immobilized to streptavidin-functionalized MAC to induce either neuronal or oligodendrocytic lineages, respectively. These bridges are tested in a rat hemisection model of SCI between T8 and T9. After eight weeks treatments including chitosan conduits result in a significant reduction in lesion area and macrophage infiltration around the lesion site (p < 0.0001). Importantly, neither immobilized IFN-γ nor PDGF-AA increased macrophage infiltration. Retrograde tracing demonstrates improved neuronal regeneration through the use of immobilized growth factors. Immunohistochemistry staining demonstrates that immobilized growth factors are effective in differentiating encapsulated cells into their anticipated lineages within the hydrogel, while qualitatively reducing glial fibrillary acid protein expression.

  3. Efficient immobilization of mushroom tyrosinase utilizing whole cells from Agaricus bisporus and its application for degradation of bisphenol A.

    Science.gov (United States)

    Kampmann, Markus; Boll, Stefan; Kossuch, Jan; Bielecki, Julia; Uhl, Stefan; Kleiner, Beatrice; Wichmann, Rolf

    2014-06-15

    A simple and efficient procedure for preparation and immobilization of tyrosinase enzyme was developed utilizing whole cells from the edible mushroom Agaricus bisporus, without the need for enzyme purification. Tyrosinase activity in the cell preparation remained constant during storage at 21 °C for at least six months. The cells were entrapped in chitosan and alginate matrix capsules and characterized with respect to their resulting tyrosinase activity. A modification of the alginate with colloidal silica enhanced the activity due to retention of both cells and tyrosinase from fractured cells, which otherwise leached from matrix capsules. The observed activity was similar to the activity that was obtained with immobilized isolated tyrosinase in the same material. Mushroom cells in water were susceptible to rapid inactivation, whereas the immobilized cells maintained 73% of their initial activity after 30 days of storage in water. Application in repeated batch experiments resulted in almost 100% conversion of endocrine disrupting bisphenol A (BPA) for 11 days, under stirring conditions, and 50-60% conversion after 20 days, without stirring under continuous usage. The results represent the longest yet reported application of immobilized tyrosinase for degradation of BPA in environmental water samples.

  4. Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone

    Institute of Scientific and Technical Information of China (English)

    Jianwei ZHEN; Qincl LIU; Jianhong GU; Yan YUAN; Xuezhong LIU; Handong WANG; Zongping LIU; Jianchun BIAN

    2012-01-01

    Abstract [Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD~o) of ZEN with tetra- zolium-based colorimetric assay (MTT assay). Comet assay was carried out to de- tect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 tJmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%. 52.00% and 64.67% under 0, 1, 5, 10 and 20 ~mo~/L o~ ZEN, respectively; the differences between the percentages of celt tail in various experimental groups had extremely significant statistical significance compared with the negative group (P〈0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60_+4.78, 57.75_+6.25, 78.97_+5.83, 100.50~6.94 and 146.83_+12.31 ~m, re- spectively; Tail DNA % in various groups was 21.29_+2.25%, 22.24_+2.43%, 31.21_+ 6.27%, 37.45_+4.33% and 60.68_+9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 ~mo~/L showed significant dif- ferences (P〈0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.

  5. Stereo-selective conversion of mandelonitrile to (R)-(−)-mandelic acid using immobilized cells of recombinant Escherichia coli

    OpenAIRE

    Pawar, Sandip V.; Meena, Vachan Singh; Kaushik, Shubhangi; Kamble, Ashwini; Kumar, Sandeep; Chisti, Yusuf; Banerjee, U. C.

    2012-01-01

    Immobilized cells of a recombinant Escherichia coli expressing nitrilase from Pseudomonas putida were used to catalyze the hydrolysis of mandelonitrile (2-hydroxy-2-phenylacetonitrile) to (R)-(−)-mandelic acid. The cells had been immobilized by entrapment in an alginate matrix. Conditions for the hydrolysis reaction were optimized in shake flasks and in a packed bed reactor. In shake flasks the best conditions for the reaction were a temperature of 40 °C, pH 8, biocatalyst bead diameter of 4....

  6. Power generation enhancement in novel microbial carbon capture cells with immobilized Chlorella vulgaris

    Science.gov (United States)

    Zhou, Minghua; He, Huanhuan; Jin, Tao; Wang, Hongyu

    2012-09-01

    With the increasing concerns for global climate change, a sustainable, efficient and renewable energy production from wastewater is imperative. In this study, a novel microbial carbon capture cell (MCC), is constructed for the first time by the introduction of immobilized microalgae (Chlorella vulgaris) into the cathode chamber of microbial fuel cells (MFCs) to fulfill the zero discharge of carbon dioxide. This process can achieve an 84.8% COD removal, and simultaneously the maximum power density can reach 2485.35 mW m-3 at a current density of 7.9 A m-3 and the Coulombic efficiency is 9.40%, which are 88% and 57.7% greater than that with suspended C. vulgaris, respectively. These enhancements in performance demonstrate the feasibility of an economical and effective approach for the simultaneous wastewater treatment, electricity generation and biodiesel production from microalgae.

  7. Immobilization of Bacillus acidocaldarius whole-cell rhodanese in polysaccharide and insolubilized gelatin gels

    Energy Technology Data Exchange (ETDEWEB)

    De Riso, L.; Alteriis, E. de; Parascandola, P. [Universita `Federico II` Napoli (Italy)]|[Istituto de Biochimica delle Proteine ed Enzimologia, Napoli (Italy); La Cara, F.; Sada, A. [Istituto di Biochimica delle Proteine ed Enzimologia, Napoli (Italy)

    1996-04-01

    The presence of rhodanese activity has been investigated in two strains of thermophilic eubacteria and two strains of extremophiles. Bacillus acidocaldarius, a thermoacidophilic eubacterium, showed the highest levels of enzyme activity. Whole cells, previously subjected to one cycle of freeze-thawing, were immobilized by entrapment in the polysaccharide matrices Ca-alginate, {kappa}-carrageenan and chitosan, and in an insolubilized gelatin gel. The results obtained with the different immobilizates in terms of activity yield, possibility of regeneration and operative stability were evaluated with the aim of setting up a continuous system. This was achieved with a system consisting of B. acidocaldarius cells entrapped in an insolubilized gelatin matrix. The latter, in the form of a thin membrane, was employed in a custom-conceived reactor operating as a plug flow reactor. 21 refs., 3 figs., 2 tabs.

  8. Production of phenolics by immobilized cells of the lichen Pseudevernia furfuracea: the role of epiphytic bacteria.

    Science.gov (United States)

    Blanch, M; Blanco, Y; Fontaniella, B; Legaz, M E; Vicente, C

    2001-06-01

    Immobilized lichen cells from the thalli of the lichen Pseudevernia furfuracea, supplied with acetate as the only source of carbon, continuously produced phenolic substances, atranorin and physodic acid, over 23 days. Epiphytic bacteria associated with the lichen thallus grew actively, probably using both acetate and reduced compounds supplied by lichen cells, since their active growth was avoided by including 10 microM 3,3'-dichlorophenyl-1,1'dimethylurea in the bath solution. Penicillin largely impeded the growth of epiphytic bacteria and decreased phenolic production, which was recovered only at the end of the experimental period, just when the bacteria started a slow, but active growth. We suggest the cooperation of epiphytic bacteria in the biosynthesis of both atranotrin and physodic acid.

  9. Optimization of Two-species Whole-cell Immobilization System Constructed with Marine-derived Fungi and Its Biological Degradation Ability

    Institute of Scientific and Technical Information of China (English)

    陈慧英; 王明霞; 沈煜斌; 姚善泾

    2014-01-01

    Mycelia pellet formed spontaneously in the process of cultivation was exploited as a biological carrier for whole-cell immobilization due to its unique structural characteristic. An innovative two-species whole-cell im-mobilization system was achieved by inoculating the marine-derived fungus Pestalotiopsis sp. J63 spores into cul-ture medium containing another fungus Penicillium janthinellum P1 pre-grown mycelia pellets for 2 days without any pretreatment. In order to evaluate the biological degradation capacity of this novel constructed immobilization system, the immobilized pellets were applied to treat paper mill effluent and decolorize dye Azure B. The use of the constructed immobilization system in the effluent resulted in successful and rapid biodegradation of numerous in-soluble fine fibers. The optimum conditions of immobilized procedure for maximum biodegradation capacity were determined using orthogonal design with biomass of P1 pellets 10 g (wet mass), concentration of J63 spore 2×109 ml-1, and immobilization time 2 d. The results demonstrate that immobilized pellets have more than 99%biodegra-dation capacity in a ten-hour treatment process. The kinetics of biodegradation fits the Michaelis-Menten equation well. Besides, the decolorization capability of immobilized pellets is more superior than that of P1 mycelia pellets. Overall, the present study offers a simple and reproducible way to construct a two-species whole-cell immobiliza-tion system for sewage treatment.

  10. Quantifying cell binding kinetics mediated by surface-bound blood type B antigen to immobilized antibodies

    Institute of Scientific and Technical Information of China (English)

    LI BaoXia; CHEN Juan; LONG Mian

    2008-01-01

    Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance (SPR)-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound biomolecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 biosensor. Human red blood cells (RBCs) presenting blood group B antigen and CM5 chip bearing immobilized anti-B monoclonal antibody (mAb) were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration,flow duration and rate, antibody-presenting level, as well as Ph value and osmotic pressure of suspending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions.These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.

  11. [Immobilization of mixed bacteria by microcapsulation for hydrogen production--a trial of pseudo "Cell Factory"].

    Science.gov (United States)

    Ma, Qianlan; Lin, Dongqiang; Yao, Shanjing

    2010-10-01

    Sodium cellulose sulfate (NaCS)/Ploy-dimethyl-dially-ammonium-chloride (PDMDAAC) microcapsules were used as a novel pseudo "Cell Factory" to immobilize mixed bacteria for hydrogen production under anaerobic conditions. Compared to free cells, the hydrogen production was increased more than 30% with NaCS/PDMDAAC microcapsules as the pseudo "Cell Factory". The biomass was increased from 1.5 g/L in free cell culture to 3.2 g/L in the pseudo "Cell Factory". This pseudo "Cell Factory" system showed the excellent stability during 15 repeated-batches. The hydrogen yield maintained 1.73-1.81 mol H2/mol glucose. The fermentation cycle was shortened from 48 h to 24 h, resulting in an increase of 198.6% in the hydrogen production rate. There were high percentage of butyric acid and acetic acid in the culture broth, which meant that the pseudo "Cell Factory" established in the present work could be used for the multi-product system.

  12. New method of immobilization of microbial cells by capture on the surface of insoluble pyridinium-type resin.

    Science.gov (United States)

    Kawabata, N; Nishimura, S; Yoshimura, T

    1990-04-25

    A new method for the immobilization of microbial cells has been developed. Whole cells of Escherichia coli with aspartase activity were immobilized by capture on the surface of cross-linked poly(N-benzyl-4-vinylpyridinium bromide) containing styrene (BVPS resin), an insoluble pyridinium-type resin. When a suspension of the bacterial cells in buffer solution was passed through a glass column containing beads of BVPS resin, the cells were captured on the resin surface and formed an immobilized cell system. A fixed-bed column reactor containing 300 mg of the bacterial cells immobilized by capture on 10 g of BVPS resin beads was used for the preparation of L-aspartic acid from ammonium fumarate. Continuous operation of tne bioreactor produced L-aspartic acid in a quantitative yield when the influent substrate concentration was 0.1M and the flow rate was 0.41-0.83 bed volumes per hour at pH 7.4-7.7 at 30 degrees C.

  13. Immobilized gellan sulfate surface for cell adhesion and multiplication: development of cell-hybrid biomaterials using self-produced fibronectin.

    Science.gov (United States)

    Miyamoto, Keiichi; Kanemoto, Akiko; Hashimoto, Kenichi; Tokita, Masayuki; Komai, Takashi

    2002-04-08

    A new concept for cell-hybrid biomaterial is proposed in which human unbilical vein endothelial cells (HUVEC) are adhered to an immobilized gellan sulfate (GS) surface. Extra domain A containing fibronectin (EDA(+)FN) released from HUVEC is necessary for cell adhesion and multiplication. The material design in this study is based on these self-released cell adhesion proteins. The interaction between GS and EDA(+)FN was evaluated using the affinity constant (KA); the value obtained was 1.03x10(8) (M(-1)). These results suggest that the adhesion of HUVEC to GS may be supported by the adhesion of EDA(+)FN to GS. We also found that this new material adheres to HUVEC, allowing the reintroduction of EDA(+)FN, which is self-produced by the cell. This material is relatively easy to produce, not requiring the usual coating of adhesion proteins in pretreatment.

  14. Electrophoresis technology

    Science.gov (United States)

    Snyder, R. S.

    1985-01-01

    A new high resolution apparatus designed for space was built as a laboratory prototype. Using a moving wall with a low zeta potential coating, the major sources of flow distortion for an electrophoretic sample stream are removed. Highly resolved fractions, however, will only be produced in space because of the sensitivity of this chamber to buoyancy-induced convection in the laboratory. The second and third flights of the McDonnell Douglas Astronautics Corporation continuous flow electrophoresis system carried samples developed at MSFC intended to evaluate the broad capabilities of free flow electrophoresis in a reduced gravity environment. Biological model materials, hemoglobin and polystyrene latex microspheres, were selected because of their past use as electrophoresis standards and as visible markers for fluid flow due to electroosmosis, spacecraft acceleration or other factors. The dependence of the separation resolution on the properties of the sample and its suspension solution was assessed.

  15. Photocurrent generation by immobilized cyanobacteria via direct electron transport in photo-bioelectrochemical cells.

    Science.gov (United States)

    Sekar, Narendran; Umasankar, Yogeswaran; Ramasamy, Ramaraja P

    2014-05-07

    Cyanobacteria possess unique and exciting features among photosynthetic microorganisms for energy conversion applications. This study focuses on production of direct electricity using a cyanobacterium called Nostoc sp. (NOS) as a photo-biocatalyst immobilized on carbon nanotubes on the anode of photo-bioelectrochemical cells. By illuminating with light (intensity 76 mW cm(-2)) the NOS immobilized on a carbon nanotube (CNT) modified electrode generated a photocurrent density of 30 mA m(-2) at 0.2 V (vs. Ag/AgCl). The contribution of different photosynthetic pigments in NOS to the light capture was analyzed and chlorophyll-a was found to be the major contributor to light capture followed by phycocyanin. Further investigation using a set of inhibitors revealed that the electrons were redirected predominantly from PSII to the CNT through the plastoquinone pool and quinol oxidase. A rudimentary design photosynthetic electrochemical cell has been constructed using NOS/CNT on the anode and laccase/CNT on the cathode as catalysts. The cell generated a maximum current density of 250 mA m(-2) and a peak power density of 35 mW m(-2) without any mediator. By the addition of 1,4-benzoquinone as a redox mediator, the electricity generation capability was significantly enhanced with a current density of 2300 mA m(-2) and a power density of 100 mW m(-2). The power densities achieved in this work are the highest among 'non-engineered' cyanobacteria based electrochemical systems reported to date.

  16. Effect of DNA damage caused by cryopreservation of spermatozoa using a modified Single cell gell electrophoresis in the freshwater catfish Pangasianodon hypophthalmus (Fowler, 1936)

    OpenAIRE

    Kuppusamy Umaa Rani; Natesan Munuswamy

    2014-01-01

    Objective: To detect the integrity of sperm DNA in the catfish Pangasianodon hypophthalmus cryopreserved with Hanks balanced salt solution, 5%-30% dimethyl acetamide (DMA) using the single-cell gel electrophoresis in order to test how sperm cryopreservation affected nuclear DNA stability. Methods: Electrophoresis was conducted for 60 min at 130 mA and 15 V. The comet images were analyzed using software CometScore 1.5, and parameters such as comet length, tail length an...

  17. Biodiesel Production: Utilization of Loofah Sponge to Immobilize Rhizopus chinensis CGMCC #3.0232 Cells as a Whole-Cell Biocatalyst.

    Science.gov (United States)

    He, Qiyang; Xia, Qianjun; Wang, Yuejiao; Li, Xun; Zhang, Yu; Hu, Bo; Wang, Fei

    2016-07-28

    Rhizopus chinensis cells immobilized on loofah (Luffa cylindrica) sponges were used to produce biodiesel via the transesterification of soybean oil. In whole-cell immobilization, loofah sponge is considered to be a superior alternative to conventional biomass carriers because of its biodegradable and renewable properties. During cell cultivation, Rhizopus chinensis mycelia can spontaneously and firmly adhere to the surface of loofah sponge particles. The optimal conditions for processing 9.65 g soybean oil at 40°C and 180 rpm using a 3:1 methanol-to-oil molar ratio were found to be 8% cell addition and 3-10% water content (depending on the oil's weight). Under optimal conditions, an over 90% methyl ester yield was achieved after the first reaction batch. The operational stability of immobilized Rhizopus chinensis cells was assayed utilizing a 1:1 methanol-to-oil molar ratio, thus resulting in a 16.5-fold increase in half-life when compared with immobilized cells of the widely studied Rhizopus oryzae. These results suggest that transesterification of vegetable oil using Rhizopus chinensis whole cells immobilized onto loofah sponge is an effective approach for biodiesel production.

  18. Analysis of Amino Acids in a Single Human Red Blood Cell by Capillary Zone Electrophoresis with Intracellular NDA—derivatization and Electrochemical Detection

    Institute of Scientific and Technical Information of China (English)

    QianDONG; XiaoLeiWANG; 等

    2002-01-01

    A novel method for determination of amino acids in individual red blood cells has been developed. In this method, the derivatization reagents (NDA and CN-) are introduced into living cells by electroporation. After completion of derivatization,the amino acids in a single cell is determined by capillary zone electrophoresis with end-column amperometric detection.

  19. Analysis of Amino Acids in a Single Human Red Blood Cell by Capillary Zone Electrophoresis with Intracellular NDA derivatization and Electrochemical Detection

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A novel method for detcrmination of amino acids in individual human red blood cells has been dcveloped. In this method, the derivatization reagents (NDA and CN-) are introduced into living cells by clcctroporation. After completion of derivatization, the amino acids in a single cell is determined by capillary zone electrophoresis with end-column ampcrometric detection.

  20. Preparation and immobilization of noble metal nanoparticles for plasmonic solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ruoli; Pitzer, Martin; Hu, DongZhi; Schaadt, Daniel M. [Institut fuer Angewandte Physik, Karlsruher Institut fuer Technologie (KIT), Karlsruhe (Germany); DFG Centrum fuer Funktionelle Nanostrukturen (CFN), KIT (Germany); Fruk, Ljiljana [DFG Centrum fuer Funktionelle Nanostrukturen (CFN), KIT (Germany)

    2011-07-01

    Thin-film solar cells are of high interest due to good electrical properties and low material consumption. Traditional thin-film cells, however, have considerable transmission losses because of the reduced absorption volume. A promising way to enhance absorption in the active layer is the light-trapping by plasmonic nanostructures. Metallic nanoparticles have in particular shown large enhancement of the photocurrent in thin-film devices. In this poster, we present preparation of Au,Ag and Pt nanoparticles by polyol method and seed mediated methods for use in plasmonic solar cells. Polyol method typically uses ethylene glycol as the solvent and reducing agent,and in seed-mediated synthesis small nanoparticle seeds are first prepared and then used to promote the growth of different shapes of nanoparticles. We particularly focus on the use of nanocubes and nanospheres for solar cell design. Following the nanoparticle preparation, a new method to immobilize particles on GaAs surfaces via covalent chemical bonds has been developed which prevents agglomerations and allows control of the surface density. Photocurrent spectra of GaAs pin solar cells with and without particles have been recorded. These measurements show the dependence of the photocurrent enhancement on particle material, shape and density.

  1. Reactor design for the ABE fermentation using cells of Clostridium acetobutylicum immobilized by adsorption onto bonechar

    Energy Technology Data Exchange (ETDEWEB)

    Qureshi, N.; Maddox, I.S.

    1988-03-07

    Cells of Clostridium acetobutylicum were immobilized by adsorption onto bonechar, and used for the production of solvents (ABE fermentation) from whey permeate. When the process was performed in packed bed reactors operated in a vertical or inclined mode, solvent productivities approximating 6kg/(m/sup 3/h) were obtained. However, the systems suffered from blockage due to excess biomass production and gas hold-up. These problems were less apparent when a partially-packed bed reactor was operated in the horizontal mode. A fluidized bed reactor proved to be the most stable of the systems investigated, and a productivity of 4.8 kg/(m/sup 3/h) was maintained over a period of 2000 h of operation. The results demonstrate that this type of reactor may have a useful future role in the ABE fermentation.

  2. Continuous acetone-butanol-ethanol production by corn stalk immobilized cells.

    Science.gov (United States)

    Zhang, Yuedong; Ma, Yujiu; Yang, Fangxiao; Zhang, Chunhui

    2009-08-01

    Corn stalk was used as a support to immobilize Clostridia beijerinckii ATCC 55025 in the fermentation process of acetone, butanol, and ethanol production. The effect of the dilution rate on solvent production was examined in a steady-state 20-day continuous flow operation. The maximum total solvent concentration of 8.99 g l(-1) was obtained at a dilution rate of 0.2 h(-1). Increasing the dilution rate between 0.2 and 1.0 h(-1) resulted in an increased solvent productivity, and the highest solvent productivity was obtained at 5.06 g l(-1) h(-1) with a dilution rate of 1 h(-1). The maximum solvent yield from glucose of 0.32 g g(-1) was observed at 0.25 h(-1). The cell adsorption and morphology change during the growth on corn stalk support were examined by the SEM.

  3. Performance of a magnetically stabilized bed reactor with immobilized yeast cells.

    Science.gov (United States)

    Ivanova, V; Hristov, J; Dobreva, E; al-Hassan, Z; Penchev, I

    1996-05-01

    This paper is focused on the possibility to apply the magnetic stabilization technique in bioprocessing. The feasibility of a continuous ethanol fermentation process with immobilized Saccharomyces cerevisiae cells in a magnetically stabilized bed (MSB) was demonstrated. The fermentation processes were carried out in an external magnetic field, transverse to the fluid flow. The flexibility to change the bed expansion owing to the independent change of the fluid flow and the field intensity (the "magnetization FIRST" mode) permitted the creation of fixed beds with different particle arrangements, which affected the bed porosity, the effective fluid-particle contact area, and the mass transfer processes on the particle-fluid interface. As a result, higher ethanol concentration, ethanol production, and glucose uptake rates than in conventional packed bed reactor were reached.

  4. SURVIVAL OF LIVER CELLS, IMMOBILIZED ON 3D-MATRIXES, IN LIVER FAILURE MODEL

    Directory of Open Access Journals (Sweden)

    M. Y. Shagidulin

    2011-01-01

    Full Text Available It was examined a new method for correction of hepatic failure by transplantation of liver support biounit (liver cells, immobilized on biocompatible and biodegradable 3D-matrixes ElastoPOB® into small intestine mesentery. It was determined that after modeling of acute hepatic failure on dogs by 65–70% liver resection and transplantation liver support biounit the restoration of disturbed biochemical indecies (such as total protein, lactate, cytolytic ensymes-ALT, AST, ALP, LDH, fibrinogen, protrombine index and others took place more rapidly on 9–14th day instead of 18th day in control. It was made a preposition about efficiency of the suggested method for correction both acute hepatic failure because even 90 days after transplantation of liver support biounit alive hepatocytes and neogenic plethoric vessels, growing through matrix were revealed. 

  5. Cell debris self-immobilized thermophilic lipase: a biocatalyst for synthesizing aliphatic polyesters.

    Science.gov (United States)

    Sun, Yang; Yang, Yan; Wang, Chenhui; Liu, Jiaming; Shi, Wei; Zhu, Xiaobo; Lu, Laijin; Li, Quanshun

    2013-05-01

    The paper explored the catalytic activity of a cell debris self-immobilized thermophilic lipase for polyester synthesis, using the ring-opening polymerization of ε-caprolactone as model. Effects of biocatalyst concentration, temperature, and reaction medium on monomer conversion and product molecular weight were systematically evaluated. The biocatalyst displayed high catalytic activity at high temperatures (70-90 °C), with 100 % monomer conversion. High monomer conversion values (>90 %) were achieved in both hydrophobic and hydrophilic solvents, and also in solvent-free system, with the exception of dichloromethane. Poly(ε-caprolactone) was obtained in 100 % monomer conversion, with a number-average molecular weight of 1,680 g/mol and a polydispersity index of 1.35 in cyclohexane at 70 °C for 72 h. Furthermore, the biocatalyst exhibited excellent operational stability, with monomer conversion values exceeding 90 % over the course of 15 batch reactions.

  6. An immobilized cell reactor with simultaneous product separation. II. Experimental reactor performance.

    Science.gov (United States)

    Dale, M C; Okos, M R; Wankat, P C

    1985-07-01

    The simultaneous separation of volatile fermentation products from product-inhibited fermentations can greatly increase the productivity of a bioreactor by reducing the product concentration in the bioreactor, as well as concentrating the product in an output stream free of cells, substrate, or other feed impurities. The Immobilized Cell Reactor-Separator (ICRS) consists of two column reactors: a cocurrent gas-liquid "enricher" followed by a countercurrent "stripper" The columns are four-phase tubular reactors consisting of (1) an inert gas phase, (2) the liquid fermentation broth, (3) the solid column internal packing, and (4) the immobilized biological catalyst or cells. The application of the ICRS to the ethanol-from-whey-lactose fermentation system has been investigated. Operation in the liquid continuous or bubble flow regime allows a high liquid holdup in the reactor and consequent long and controllable liquid residence time but results in a high gas phase pressure drop over the length of the reactor and low gas flow rates. Operation in the gas continuous regime gives high gas flow rates and low pressure drop but also results in short liquid residence time and incomplete column wetting at low liquid loading rates using conventional gas-liquid column packings. Using cells absorbed to conventional ceramic column packing (0.25-in. Intalox saddles), it was found that a good reaction could be obtained in the liquid continuous mode, but little separation, while in the gas continuous mode there was little reaction but good separation. Using cells sorbed to an absorbant matrix allowed operation in the gas continuous regime with a liquid holdup of up to 30% of the total reactor volume. Good reaction rates and product separation were obtained using this matrix. High reaction rates were obtained due to high density cell loading in the reactor. A dry cell density of up to 92 g/L reactor was obtained in the enricher. The enricher ethanol productivity ranged from 50 to 160

  7. Determination of the Mutagenicity Potential of Supermint Herbal Medicine by Single Cell Gel Electrophoresis in Rat Hepatocytes

    Directory of Open Access Journals (Sweden)

    Zivar Amanpour

    2012-08-01

    Full Text Available Purpose: The increasing use of herbal drugs and their easy availability have necessitated the use of mutagenicity test to analyze their toxicity and safety. The aim of this study was to evaluate the genotoxicity of Supermint herbal medicine in DNA breakage of rat hepatocytes in comparison with sodium dichromate by single cell gel electrophoresis technique or comet assay. Methods: Hepatocytes were prepared from male wistar rats and were counted and kept in a bioreactor for 30 minutes. Then cells were exposed to the Supermint herbal medicine at doses of 125, 250 and 500 μl/ml. Buffer 4 (incubation buffer and sodium dichromate were used as negative and positive control for one hour respectively. Then cell suspension with low melting point agarose were put on precoated slides and covered with agarose gel. Then lysing, electrophoresis, neutralization and staining were carried out. Finally the slides were analyzed with fluorescence microscope. The parameter under this analysis was the type of migration which was determined according to Kobayashi pattern. Results: With increased dose of Supermint herbal medicine the DNA damage was slightly increased (P<0001. Conlusion: In overall compared to the positive control significant differences is observed which convinced that the crude extract of Supermint in vitro did not have mutagenic effect.

  8. Proliferation of endothelial cells on surface-immobilized albumin-heparin conjugate loaded with basic fibroblast growth factor

    NARCIS (Netherlands)

    Bos, Gert W.; Scharenborg, Nicole M.; Poot, André A.; Engbers, Gerard H.M.; Beugeling, Tom; Aken, van Willem G.; Feijen, Jan

    1999-01-01

    Seeding of endothelial cells (ECs) on the luminal surface of small-diameter vascular grafts is a promising method to avoid occlusion of these prostheses. Immobilization of basic fibroblast growth factor (bFGF) to substrates used to coat or fill porous prostheses may enhance the formation of a conflu

  9. Optimization of ethanol production from carob pod extract using immobilized Saccharomyces cerevisiae cells in a stirred tank bioreactor.

    Science.gov (United States)

    Ercan, Yatmaz; Irfan, Turhan; Mustafa, Karhan

    2013-05-01

    In this study, optimization of ethanol production from carob pod extract was carried out by immobilized Saccharomyces cerevisiae. Results showed that Ca-alginate concentration and the amount of immobilized cells had significant effects on yield. Optimum conditions for ethanol fermentation were determined to be 2% Ca-alginate concentration, 150 rpm agitation rate, 5% yeast cells entrapped in beads and pH 5.5. After validation experiments; ethanol concentration, yield, production rate and sugar utilization rate were respectively 40.10 g/L, 46.32%, 3.19 g/L/h and 90.66%; and the fermentation time was decreased to 24 h. In addition, the immobilized cells were shown to be reusable for five cycles, though a decrease in yield was observed. Finally, carob pod extract was used for ethanol fermentation by controlled and uncontrolled pH without any enrichment, and the results suggest that carob extract can be utilized effectively by immobilized-cell fermentation without the use of enrichments to facilitate yeast growth.

  10. Site-protected fixation and immobilization of Escherichia coli cells displaying surface-anchored beta-lactamase.

    Science.gov (United States)

    Freeman, A; Abramov, S; Georgiou, G

    1999-01-20

    Bacteria displaying heterologous receptors or enzymes on their surface hold great potential as whole-cell adsorbents and biocatalysts, respectively. For industrial applications, such surface-engineered cells need to be killed and chemically fixed to prevent disintegration and leakage of the displayed proteins under process conditions. It is also highly desirable to couple the chemically stabilized cells onto a solid support matrix for additional mechanical stability, flexibility in reactor choice, and easy separation from processed medium. Recently, we described the development of a readily scalable methodology for cell killing, fixation, and outer membrane stabilization via glutaraldehyde fixation followed by secondary crosslinking (Freeman, A., Abramov, S. and Georgiou, G. 1996. Biotechnol. Bioeng. 52: 625-630). Glutaraldehyde treatment was also found, however, to reduce the specific activity of a model enzyme, beta-lactamase displayed on the surface of E. coli. Here, we show that crosslinking carried out in the presence of beta-lactamase inhibitors, namely phenyl boronic acid or sodium borate, protects the active site from chemical modification resulting in up to threefold higher specific activities without affecting the cell-stabilizing effect of the glutaraldehyde treatment. To prepare an immobilized whole cell biocatalyst, residual unreacted surface aldehyde groups were employed to immobilize covalently the fixed bacteria onto chitosan-coated cellulose powder. The binding of the bacteria onto chitosan-coated cellulose was quantitative up to cell loading of 83 mg dry cell weight/g of support. Cell immobilization did not introduce mass transfer limitations and created only a modest reduction in Vmax. Thus, chemical crosslinking, affected in presence of reversible active-site inhibitors and coupled with cell immobilization on chitosan-coated cellulose represents a widely useful methodology for the process application of recombinant bacteria displaying surface

  11. Immobilization of Galphimia glauca plant cell suspensions for the production of enhanced amounts of Galphimine-B.

    Science.gov (United States)

    Osuna, Lidia; Moyano, Elisabeth; Mangas, Susana; Bonfill, Mercè; Cusidó, Rosa M; Piñol, M Teresa; Zamilpa, Alejandro; Tortoriello, Jaime; Palazón, Javier

    2008-01-01

    We tested the capacity of Galphimia glauca cells to produce galphimine-B (G-B) when under the effects of a two-stage culture system: cell immobilization in Ca2+-alginate beads and culture scale-up from shake-flask to two different types of bioreactor (stirred and airlift). In the shake-flask culture, using optimum media for cell growth (first stage) and G-B production (second stage), the G-B yield was similar in both immobilised and free cells. However, while the free cells accumulated G-B within cytoplasmatic compartments, where it could not be recovered without cell disruption, immobilized cells excreted up to 100 % of the G-B produced. Immobilized cells grown in bioreactors running for 14 days with growth medium and an additional 26 days with production medium in batch mode showed a high G-B yield. The stirred bioreactor was the most efficient with a G-B content in the culture medium of 1381 microg.L (-1) at day 24 of culture.

  12. Study on the mechanism of Bioelectric Recognition Assay: evidence for immobilized cell membrane interactions with viral fragments.

    Science.gov (United States)

    Kintzios, S; Bem, F; Mangana, O; Nomikou, K; Markoulatos, P; Alexandropoulos, N; Fasseas, C; Arakelyan, V; Petrou, A-L; Soukouli, K; Moschopoulou, G; Yialouris, C; Simonian, A

    2004-11-01

    The Bioelectric Recognition Assay (BERA) is a whole-cell based biosensing system that detects the electric response of cultured cells, suspended in a gel matrix, to various ligands, which bind to the cell and/or affect its physiology. Previous studies have demonstrated the potential application of this method for rapid, inexpensive detection of viruses in a crude sample. However, the understanding, so far, of the fundamental processes that take place during cell-virus interactions within the probe has been rather limited. In the present study, we combined electrophysiological and fluorescence microscopical assays, so that we can prove that animal and plant cells immobilized in BERA sensors respond to different viruses primarily by changing their membrane potential. The response of immobilized cells against different viruses did not depend on the virus ability to penetrate the cell, but was modified after binding each virus to a virus-specific antibody or removal of its coat protein after treatment with a protease. Consequently, we were able to assay the presence of a virus in its complete form or fragments thereof. Combination of immunological recognition with the electrophysiological response of immobilized cells allows for a considerable increase of the specificity of the BERA biosensory assay. In addition, rather than simply detect the presence of a protein or genomic sequence, the method can help gain information on the bioactivity of a virus.

  13. Immobilization free electrochemical biosensor for folate receptor in cancer cells based on terminal protection.

    Science.gov (United States)

    Ni, Jiancong; Wang, Qingxiang; Yang, Weiqiang; Zhao, Mengmeng; Zhang, Ying; Guo, Longhua; Qiu, Bin; Lin, Zhenyu; Yang, Huang-Hao

    2016-12-15

    The determination of folate receptor (FR) that over expressed in vast quantity of cancerous cells frequently is significant for the clinical diagnosis and treatment of cancers. Many DNA-based electrochemical biosensors have been developed for FR detection with high selectivity and sensitivity, but most of them need complicated immobilization of DNA on the electrode surface firstly, which is tedious and therefore results in the poor reproducibility. In this study, a simple, sensitive, and selective electrochemical FR biosensor in cancer cells has been proposed, which combines the advantages of the convenient immobilization-free homogeneous indium tin oxide (ITO)-based electrochemical detection strategy and the high selectivity of the terminal protection of small molecule linked DNA. The small molecule of folic acid (FA) and an electroactive molecule of ferrocence (Fc) were tethered to 3'- and 5'-end of an arbitrary single-stranded DNA (ssDNA), respectively, forming the FA-ssDNA-Fc complex. In the absence of the target FR, the FA-ssDNA-Fc was degraded by exonuclease I (Exo I) from 3'-end and produced a free Fc, diffusing freely to the ITO electrode surface and resulting in strong electrochemical signal. When the target FR was present, the FA-ssDNA-Fc was bound to FR through specific interaction with FA anchored at the 3'-end, effectively protecting the ssDNA strand from hydrolysis by Exo I. The FR-FA-ssDNA-Fc could not diffuse easily to the negatively charged ITO electrode surface due to the electrostatic repulsion between the DNA strand and the negatively charged ITO electrode, so electrochemical signal reduced. The decreased electrochemical signal has a linear relationship with the logarithm of FR concentration in range of 10fM to 10nM with a detection limit of 3.8fM (S/N=3). The proposed biosensor has been applied to detect FR in HeLa cancer cells, and the decreased electrochemical signal has a linear relationship with the logarithm of cell concentration ranging

  14. Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2012-06-01

    Full Text Available Abstract Background Microbial laccases are highly useful in textile effluent dye biodegradation. However, the bioavailability of cellularly expressed or purified laccases in continuous operations is usually limited by mass transfer impediment or enzyme regeneration difficulty. Therefore, this study develops a regenerable bacterial surface-displaying system for industrial synthetic dye decolorization, and evaluates its effects on independent and continuous operations. Results A bacterial laccase (WlacD was engineered onto the cell surface of the solvent-tolerant bacterium Pseudomonas putida to construct a whole-cell biocatalyst. Ice nucleation protein (InaQ anchor was employed, and the ability of 1 to 3 tandemly aligned N-terminal repeats to direct WlacD display were compared. Immobilized WlacD was determined to be surface-displayed in functional form using Western blot analysis, immunofluorescence microscopy, flow cytometry, and whole-cell enzymatic activity assay. Engineered P. putida cells were then applied to decolorize the anthraquinone dye Acid Green (AG 25 and diazo-dye Acid Red (AR 18. The results showed that decolorization of both dyes is Cu2+- and mediator-independent, with an optimum temperature of 35°C and pH of 3.0, and can be stably performed across a temperature range of 15°C to 45°C. A high activity toward AG25 (1 g/l with relative decolorization values of 91.2% (3 h and 97.1% (18 h, as well as high activity to AR18 (1 g/l by 80.5% (3 h and 89.0% (18 h, was recorded. The engineered system exhibited a comparably high activity compared with those of separate dyes in a continuous three-round shake-flask decolorization of AG25/AR18 mixed dye (each 1 g/l. No significant decline in decolorization efficacy was noted during first two-rounds but reaction equilibriums were elongated, and the residual laccase activity eventually decreased to low levels. However, the decolorizing capacity of the system was easily retrieved

  15. Detection of connexins in liver cells using sodiumdodecylsulfate polyacrylamide gel electrophoresis and immunoblot analysis

    Science.gov (United States)

    Willebrords, Joost; Maes, Michaël; Yanguas, Sara Crespo; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Summary Since connexin expression is partly regulated at the protein level, immunoblot analysis represents a frequently addressed technique in the connexin research field. The present chapter describes the set-up of an immunoblot procedure, including protein extraction and quantification from biological samples, gel electrophoresis, protein transfer and immunoblotting, which is optimized for analysis of connexins in liver tissue. In essence, proteins are separated on a polyacrylamide gel using sodiumdodecylsulfate followed by transfer of proteins on a nitrocellulose membrane. The latter allows specific detection of connexins with antibodies combined with revelation through enhanced chemiluminescence. PMID:27207285

  16. Immobilization of gold nanoparticles on cell culture surfaces for safe and enhanced gold nanoparticle-mediated laser transfection

    Science.gov (United States)

    Kalies, Stefan; Heinemann, Dag; Schomaker, Markus; Gentemann, Lara; Meyer, Heiko; Ripken, Tammo

    2014-07-01

    In comparison to standard transfection methods, gold nanoparticle-mediated laser transfection has proven to be a versatile alternative. This is based on its minor influence on cell viability and its high efficiency, especially for the delivery of small molecules like small interfering RNA. However, in order to transfer it to routine usage, a safety aspect is of major concern: The avoidance of nanoparticle uptake by the cells is desired. The immobilization of the gold nanoparticles on cell culture surfaces can address this issue. In this study, we achieved this by silanization of the appropriate surfaces and the binding of gold nanoparticles to them. Comparable perforation efficiencies to the previous approaches of gold nanoparticle-mediated laser transfection with free gold nanoparticles are demonstrated. The uptake of the immobilized particles by the cells is unlikely. Consequently, these investigations offer the possibility of bringing gold nanoparticle-mediated laser transfection closer to routine usage.

  17. Immobilization of gold nanoparticles on cell culture surfaces for safe and enhanced gold nanoparticle-mediated laser transfection.

    Science.gov (United States)

    Kalies, Stefan; Heinemann, Dag; Schomaker, Markus; Gentemann, Lara; Meyer, Heiko; Ripken, Tammo

    2014-01-01

    In comparison to standard transfection methods, gold nanoparticle-mediated laser transfection has proven to be a versatile alternative. This is based on its minor influence on cell viability and its high efficiency, especially for the delivery of small molecules like small interfering RNA. However, in order to transfer it to routine usage, a safety aspect is of major concern: The avoidance of nanoparticle uptake by the cells is desired. The immobilization of the gold nanoparticles on cell culture surfaces can address this issue. In this study, we achieved this by silanization of the appropriate surfaces and the binding of gold nanoparticles to them. Comparable perforation efficiencies to the previous approaches of gold nanoparticle-mediated laser transfection with free gold nanoparticles are demonstrated. The uptake of the immobilized particles by the cells is unlikely. Consequently, these investigations offer the possibility of bringing gold nanoparticle-mediated laser transfection closer to routine usage.

  18. A novel ethanol/oxygen microfluidic fuel cell with enzymes immobilized onto cantilevered porous electrodes

    Science.gov (United States)

    Desmaële, D.; Nguyen-Boisse, T. T.; Renaud, L.; Tingry, S.

    2016-11-01

    This paper introduces a novel design of membraneless microfluidic biofuel cell that incorporates three-dimensional porous electrodes containing immobilized enzymes to catalyze redox reactions occurring in the presence of ethanol/O2 co-laminar flows. In order to maximize the penetration depth of the reactants inside the porous medium, we report on the preliminary evaluation of cantilevered bioelectrodes, namely the fibrous electrodes protrude along the internal walls of the miniature electrochemical chamber. As a first proof-of-concept, we demonstrate the integration of a bioanode and a biocathode into a lamination-based microfluidic cell fabricated via rapid prototyping. With enzymes deposited into the fibrous structure of 25 mm long, 1 mm wide and 0.11 mm thick carbon paper electrodes, the volumetric power density reached 1.25 mW cm-3 at 0.43 V under a flow rate of 50 μL min-1. An advantage of the presented microfluidic biofuel cell is that it can be adapted to include a larger active electrode volume via the vertical stacking of multiple thin bioelectrodes. We therefore envision that our design would be amenable to reach the level of net power required to supply energy to a plurality of low-consumption electronic devices.

  19. Magnetic immobilization of Bacillus subtilis natto cells for menaquinone-7 fermentation.

    Science.gov (United States)

    Ebrahiminezhad, Alireza; Varma, Vikas; Yang, Shuyi; Berenjian, Aydin

    2016-01-01

    Production of menaquinone-7 (MK-7) by Bacillus subtilis natto is associated with major drawbacks. To address the current challenges in MK-7 fermentation, studying the effect of magnetic nanoparticles on the bacterial cells can open up a new domain for intensified bioprocesses. This article introduces the new concept of application of iron oxide nanoparticles (IONs) as a pioneer tool for MK-7 process intensification. In this order, IONs with the average size of 11 nm were successfully fabricated and characterized for possible in situ removal of target substances from the fermentation media. The prepared particles were used for decoration and immobilization of B. subtilis natto cells. Presence of iron oxide nanoparticles significantly enhanced the MK-7 specific yield (15 %) as compared to the control samples. In addition, fabricated IONs showed a promising ability for in situ recovery of bacterial cells from the fermentation media with more than 95 % capture efficiency. Based on the results, IONs can be implemented successfully as a novel tool for MK-7 production. This study provides a considerable interest for industrial application of magnetic nanoparticles and their future role in designing an intensified biological process.

  20. Immobilization of Microbial Cells for Alcoholic and Malolactic Fermentation of Wine and Cider

    Science.gov (United States)

    Kourkoutas, Yiannis; Manojlović, Verica; Nedović, Viktor A.

    Wine- or cider-making is highly associated with biotechnology owing to the traditional nature of must fermentation.. Nowadays, there have been considerable developments in wine- or cider-making techniques affecting all phases of wine or cider production, but more importantly, the fermentation process. It is well-known that the transformation of grape must by microbial activity results in the production of wine, and the fermentation of apples (or sometimes pears) in the production of cider. In this process, a variety of compounds affecting the organoleptic profile of wine or cider are synthesized. It is also common sense that in wine- or cider-making, the main objective is to achieve an adequate quality of the product. The technological progress and the improved quality of the wines or ciders have been associated with the control of technical parameters. Herein, cell immobilization offers numerous advantages, such as enhanced fermentation productivity, ability for cell recycling, application of continuous configurations, enhanced cell stability and viability, and improvement of quality (Margaritis and Merchant 1984; Stewart and Russel 1986; Kourkoutas et al. 2004a).

  1. Two-dimensional gel electrophoresis data for proteomic profiling of Sporothrix yeast cells

    Directory of Open Access Journals (Sweden)

    Anderson Messias Rodrigues

    2015-03-01

    Full Text Available Sporotrichosis is a chronic infection of the skin and subcutaneous tissues of human and other mammals caused by a complex of cryptic dimorphic fungi in the plant-associated order Ophiostomatales. With major differences between routes of transmission, Sporothrix infections are emerging as new threat in tropical and subtropical areas, particularly in form of outbreaks. The mechanisms underlying the pathogenesis and invasion of Sporothrix spp. are still poorly understood and many virulence factors remain unidentified. In this scenario, a global analysis of proteins expressed by clinical Sporothrix species combined with the identification of seroreactive proteins is overdue. Optimization of sample preparation and electrophoresis conditions are key steps toward reproducibility of gel-based proteomics assays. We provide the data generated using an efficient protocol of protein extraction for rapid and large-scale proteome analysis using two-dimensional gel electrophoresis. The protocol was established and optimized for pathogenic and non-pathogenic Sporothrix spp. including Sporothrix brasiliensis (CBS 132990, Sporothrix schenckii sensu stricto (CBS 132974, Sporothrix globosa (CBS 132922, and Sporothrix mexicana (CBS 120341. The data, supplied in this article, are related to the research article entitled “Immunoproteomic analysis reveals a convergent humoral response signature in the Sporothrix schenckii complex” (Rodrigues et al., 2014 [1].

  2. Evaluation of Osteoblast-Like Cell Viability and Differentiation on the Gly-Arg-Gly-Asp-Ser Peptide Immobilized Titanium Dioxide Nanotube via Chemical Grafting.

    Science.gov (United States)

    Kim, Ga-Hyun; Kim, Il-Shin; Park, Sang-Won; Lee, Kwangmin; Yun, Kwi-Dug; Kim, Hyun-Seung; Oh, Gye-Jeong; Ji, Min-Kyung; Lim, Hyun-Pil

    2016-02-01

    This study examined the effect of the immobilization of the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide on titanium dioxide (TiO2) nanotube via chemical grafting on osteoblast-like cell (MG-63) viability and differentiation. The specimens were divided into two groups; TiO2 nanotubes and GRGDS-immobilized TiO2 nanotubes. The surface characteristics of GRGDS-immobilized TiO2 nanotubes were observed by using X-ray photoelectron spectroscopy (XPS) and a field emission scanning electron microscope (FE-SEM). The morphology of cells on specimens was observed by FE-SEM after 2 hr and 24 hr. The level of cell viability was investigated via a tetrazolium (XTT) assay after 2 and 4 days. Alkaline phosphatase (ALP) activity was evaluated to measure the cell differentiation after 4 and 7 days. The presence of nitrogen up-regulation or C==O carbons con- firmed that TiO2 nanotubes were immobilized with GRGDS peptides. Cell adhesion was enhanced on the GRGDS-immobilized TiO2 nanotubes compared to TiO2 nanotubes. Furthermore, significantly increased cell spreading and proliferation were observed with the cells grown on GRGDS-immobilized TiO2 nanotubes (P nanotubes and TiO2 nanotubes. These results suggest that the GRGDS-immobilized TiO2 nanotubes might be effective in improving the osseointegration of dental implants.

  3. Development of thrombus-resistant and cell compatible crimped polyethylene terephthalate cardiovascular grafts using surface co-immobilized heparin and collagen

    Energy Technology Data Exchange (ETDEWEB)

    Al Meslmani, Bassam, E-mail: almeslmanib@yahoo.com [Department of Pharmaceutical Technology and Biopharmaceutics, Marburg University, Ketzerbach 63, 35037 Marburg (Germany); Mahmoud, Gihan, E-mail: mahmoudg@staff.uni-marburg.de [Department of Pharmaceutical Technology and Biopharmaceutics, Marburg University, Ketzerbach 63, 35037 Marburg (Germany); Department of Pharmaceutics and Industrial Pharmacy, Helwan University, Ain Helwan, 11795 Cairo (Egypt); Strehlow, Boris, E-mail: strehlo4@staff.uni-marburg.de [Department of Pharmaceutical Technology and Biopharmaceutics, Marburg University, Ketzerbach 63, 35037 Marburg (Germany); Mohr, Eva, E-mail: mohr@staff.uni-marburg.de [Department of Pharmaceutical Technology and Biopharmaceutics, Marburg University, Ketzerbach 63, 35037 Marburg (Germany); Leichtweiß, Thomas, E-mail: Thomas.leichtweiss@phys.chemie.uni-giessen.de [Institute of Physical Chemistry, Justus-Liebig-University, Heinrich-Buff-Ring 58, 35392 Giessen (Germany); Bakowsky, Udo, E-mail: ubakowsky@aol.com [Department of Pharmaceutical Technology and Biopharmaceutics, Marburg University, Ketzerbach 63, 35037 Marburg (Germany)

    2014-10-01

    Short-term patency of polyethylene terephthalate (PET) cardiovascular grafts is determined mainly by the inherent thrombogenicity and improper endothelialization following grafts implantation. The aim of the present study was to immobilize heparin to develop thrombus resistant grafts. Additionally, collagen was co-immobilized to enhance the host cell compatibility. The synthetic woven and knitted forms of crimped PET grafts were surface modified by Denier reduction to produce functional carboxyl groups. The produced groups were used as anchor sites for covalent immobilization of heparin or co-immobilization of heparin/collagen by the end-point method. The modified surface was characterized using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The biological activity of immobilized molecules was investigated in vitro using direct blood coagulation test, and “platelet deposition under flow condition. Furthermore, the biocompatibility of modified grafts with host cells was assessed using L929 cell as model. All modified grafts showed significant resistance against fibrin and clot formation. The number of deposited platelets on heparin-immobilized woven and knitted grafts obviously decreased by 3 fold and 2.8 fold per unit surface area respectively, while the heparin/collagen co-immobilized grafts showed only a decrease by 1.7 and 1.8 fold compared to unmodified PET. Heparin-immobilized grafts reported no significant effect on L929 cells adhesion and growth (P > 0.05), conversely, collagen co-immobilization considerably increased cell adhesion almost ∼ 1.3 fold and 2 fold per unit surface area for woven and knitted grafts respectively. Our results emphasize that immobilization of heparin minimized the inherent thrombogenicity of the PET grafts. The simultaneous co-immobilization of collagen supported host cell adhesion and growth required for the grafts biocompatibility. - Highlight: • Heparin and collagen were co-immobilized on

  4. Design and experimental verification of low-voltage two-dimensional CMOS electrophoresis platform with 32 × 32 sample/hold cell array

    Science.gov (United States)

    Yamaji, Yuuki; Niitsu, Kiichi; Nakazato, Kazuo

    2016-03-01

    Electrophoresis is widely used in biomedical applications. However, conventional (centimeter-order) electrophoresis requires a high-voltage power supply, which is not suitable for point-of-care testing (POCT). Electrophoresis is driven by electric fields, and miniaturization (from the centimeter order to the micrometer order) is effective for low-voltage operation. A CMOS platform is a cost-competitive and promising candidate for miniaturization and enables the integration of biomolecule manipulation by electrophoresis and its electrochemical sensing. These features will contribute to the development of a biochemical analyzer called the micro-total analysis system (µ-TAS). To realize a truly portable electrophoresis system, we present the design and experimental verification of a low-voltage (<1 V), two-dimensional CMOS electrophoresis platform with 32 × 32 sample/hold cell array. Experimental results showed successful constant voltage outputs to each electrode. By miniaturizing the electrode structure to a 60 µm pitch, we achieved sufficient electric field strength even at low voltages.

  5. Properties of catechol 1,2-dioxygenase in the cell free extract and immobilized extract of Mycobacterium fortuitum

    Directory of Open Access Journals (Sweden)

    A.S. Silva

    2013-01-01

    Full Text Available Polycyclic aromatic hydrocarbons (PAH are carcinogenic compounds which contaminate water and soil, and the enzymes can be used for bioremediation of these environments. This study aimed to evaluate some environmental conditions that affect the production and activity of the catechol 1,2-dioxygenase (C12O by Mycobacterium fortuitum in the cell free and immobilized extract in sodium alginate. The bacterium was grown in mineral medium and LB broth containing 250 mg L-1 of anthracene (PAH. The optimum conditions of pH (4.0-9.0, temperature (5-70 ºC, reaction time (10-90 min and the effect of ions in the enzyme activity were determined. The Mycobacterium cultivated in LB shown higher growth and the C12O activity was two-fold higher to that in the mineral medium. To both extracts the highest enzyme activity was at pH 8.0, however, the immobilized extract promoted the increase in the C12O activity in a pH range between 4.0 and 8.5. The immobilized extract increased the enzymatic activity time and showed the highest C12O activity at 45 ºC, 20 ºC higher than the greatest temperature in the cell free extract. The enzyme activity in both extracts was stimulated by Fe3+, Hg2+ and Mn2+ and inhibited by NH4+ and Cu2+, but the immobilization protected the enzyme against the deleterious effects of K+ and Mg2+ in tested concentrations. The catechol 1,2-dioxygenase of Mycobacterium fortuitum in the immobilized extract has greater stability to the variations of pH, temperature and reaction time, and show higher activity in presence of ions, comparing to the cell free extract.

  6. Biodegradation of drotaverine hydrochloride by free and immobilized cells of Rhodococcus rhodochrous IEGM 608.

    Science.gov (United States)

    Ivshina, I B; Vikhareva, E V; Richkova, M I; Mukhutdinova, A N; Karpenko, Ju N

    2012-10-01

    Drotaverine [1-(3,4-diethoxybenzylidene)-6,7-diethoxy-1,2,3,4-tetrahydroisoquinoline] hydrochloride, an antispasmodic drug derived from benzylisoquinoline was evaluated for its biodegradability using a bacterial strain Rhodococcus rhodochrous IEGM 608. The experiments were performed under aerobic conditions with rhodococci cultures able to degrade drotaverine. In the presence of glucose, the removal efficiency of drotaverine by free Rhodoccocus cells pre-grown with isoquinoline was above 80 % (200 mg/l, initial concentration) after 25 days. Rhodococcus immobilization on hydrophobized sawdust enhanced the biodegradation process, with the most marked drotaverine loss being observed during the first 5 days of fermentation. High metabolic activity of rhodococcal cells towards drotaverine was confirmed respirometrically. GC-MS analysis of transformation products resulting from drotaverine biodegradation revealed 3,4-diethoxybenzoic acid, 3,4-diethoxybenzaldehyde and 3,4-diethoxybenzoic acid ethyl ester which were detected in the culture medium until drotaverine completely disappeared. Based on these major and other minor metabolites, putative pathways for drotaverine biodegradation were proposed. The obtained data broadened the spectrum of organic xenobiotics oxidized by Rhodoccocus bacteria and proved their potential in decontamination of natural ecosystems from pharma pollutants.

  7. Comparative investigations of T cell receptor gamma gene rearrangements in frozen and formalin-fixed paraffin wax-embedded tissues by capillary electrophoresis

    DEFF Research Database (Denmark)

    Christensen, M; Funder, A D; Bendix, K;

    2006-01-01

    AIM: To compare clonal T cell receptor gamma (TCRgamma) gene rearrangements in frozen and formalin-fixed paraffin wax-embedded (FFPE) tissue, using capillary electrophoresis for use in diagnostics, as T cell lymphomas may be difficult to diagnose by conventional methods. METHODS: The DNA for PCR......% for patient specimens and the specificity 100%. The junctional region between the Vgamma and Jgamma segments was specific for each patient. CONCLUSIONS: Capillary electrophoresis of PCR products from frozen and FFPE tissue is suitable for detecting clonal TCRgamma gene rearrangements. It is important, however...

  8. Single-step immobilization of cell adhesive peptides on a variety of biomaterial substrates via tyrosine oxidation with copper catalyst and hydrogen peroxide.

    Science.gov (United States)

    Kakinoki, Sachiro; Yamaoka, Tetsuji

    2015-04-15

    Immobilization of biologically active peptides which were isolated from extracellular matrix proteins is a powerful strategy for the design and functionalization of biomaterial substrates. However, the method of peptide immobilization was restricted, that is, peptide is often immobilized through the reactive groups inherent in substrates with multistep reactions. Here, we report a single-step immobilization of fibronectin-derived cell adhesive peptide (Arg-Glu-Asp-Val; REDV) onto polymer materials by use of tyrosine oxidation with copper catalyst and hydrogen peroxide. REDV peptide was successfully immobilized on tissue culture polystyrene, poly(ethylene terephthalate), poly(vinyl chloride), expanded-poly(tetrafluoroethylene), and poly(l-lactic acid), resulting in enhanced adhesion of human umbilical vein endothelial cells. This method is a single-step reaction under very mild conditions and is available for the biological functionalization of various medical devices.

  9. Removal of lead in wastewater by immobilized inactivated cells of Rhizopus oligosporus

    Institute of Scientific and Technical Information of China (English)

    于霞; 柴立元; 闵小波

    2003-01-01

    A novel technology for lead removal with nonliving Rhizopus oligosporus immobilized in calcium alginate was studied. The results show that the main influencing factors include pH value and interfering cations. pH value has different effects on biosorption of various heavy metals and lead adsorption can be proceeded by controlling pH value in a range of 2-5; interfering cations especially Cu( Ⅱ ) can make the adsorption amount of Pb( Ⅱ ) decrease by immobilized Rhizopus oligosporus. Desorption efficiency of different eluants and kinetics were investigated. Citrate the reaction equilibrium reaches 3 h. Immobilized biomass keeps high lead biosorption capacity after five cycles of regeneration.

  10. Stabilization of k-carrageenan gel with polymeric amines: use of immobilized cells as biocatalysts at elevated temperatures

    Energy Technology Data Exchange (ETDEWEB)

    Chao, K.C.; Haugen, M.M.; Royer, G.P.

    1986-09-01

    Spherical beads of kappa-carrageenan containing entrapped cells were prepared in a two-step process. First, the beads were formed by dispersing a warm carrageenan cell suspension into stirring oil. After cooling (gelation) the beads were cured by treatment with amines. Ten amines of various sizes and structures were tested. The mechanical strength and the applicability of amine-treated gels as immobilization matrices was evaluated. The results of critical compression tests indicate that linear and branched polyethylenimines (PEI) are both good curing agents. PEI-treated carrageenan beds also exhibited superior resistance to heat and abrasion. Furthermore, PEI polymers were demonstrated to be effective in stabilizing the lactase activity of the free and immobilized Bacillus stearothermophilus cells. The immobilized cell preparations of Saccharomyces cerevisiae, B. stearothermophilus, and Flavobacterium sp. were treated with branched PEI and evaluated for the activity of invertase (EC 3.2.1.26), lactase (EC 3.2.1.23), and glucose isomerase (EC 5.3.1.18), respectively, in a packed bed reactor at 60 degrees C. The apparent half-lives were 108, 39, and 64 days, respectively.

  11. Evaluation of radio-induced DNA damage and their repair in human lymphocytes by comet assay or single cell gel electrophoresis; Avaliacao do dano radioinduzido no DNA e reparo em linfocitos humanos pelo metodo do cometa (single cell gel electrophoresis)

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, Patricia A. do; Suzuki, Miriam F.; Okazaki, Kayo [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil)

    1997-12-01

    The comet assay, also called single cell gel electrophoresis technique, permits to evaluate quantitatively DNA breakage induced by chemical and physical agents at the level of the single cell. The present paper refers to the construction of dose-response curves to DNA damage and repair studies in human peripheral lymphocytes, utilizing the comet assay for the radiosensitivity analysis. So, the blood samples were obtained from healthy donors (40-50 year old), irradiated in a {sup 60} Co source (GAMMACEL 220) with doses of 0.17, 0.25, 0.57, 1.10, 2.12 and 4.22 Gy (0.59 Gy/min.) and processed 1 and 24 hours after the exposition. Results obtained showed a increase in the total lenght of comet (DNA migration) as a function of radiation dose in samples processed 1 and 24 hours after the treatment. The DNA lesion in irradiated lymphocytes with 4.22 Gy (means value of 101.4 {mu}m) were 3.4 times higher than in the untreated lymphocytes (mean value of 30 {mu}m) instead of 24 hours after the irradiation were 1.5 times higher (mean value of 46.3 {mu}m). This reduction on DNA repair occurred in these cells. It was also possible visualized the presence of subpopulations of the cells with different sensitivity and repair capacity to ionizing radiation in these donors. (author). 8 refs., 3 figs.

  12. 44. Study the level of DNA breakage in workers exposed to styrene by single cell gel electrophoresis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: Study the level of DNA breakage in workers exposed to styrene. Methods: 35 workers aging from 18 to 40 exposed to styrene half a year above were observed as exposed group, in the mean time, 57 workers in the same district who hadn't been exposed to known genotoxicant were selected as control. Bloods of them were sampled and DNA lesions were detected by single cell gel electrophoresis. Results: Compared with control, the ratio between the length of Comet tail and the total length of Comet in exposed group significantly increased, especially it raised following the styrene concentration exposed, but it was not different among different working age groups. Conclusions: DNA is damaged by styrene, and it appears as dose-response relationship.

  13. Immobilization of Electroporated Cells for Fabrication of Cellular Biosensors: Physiological Effects of the Shape of Calcium Alginate Matrices and Foetal Calf Serum

    Directory of Open Access Journals (Sweden)

    Nikos Katsanakis

    2009-01-01

    Full Text Available In order to investigate the physiological effect of transfected cell immobilization in calcium alginate gels, we immobilized electroporated Vero cells in gels shaped either as spherical beads or as thin membrane layers. In addition, we investigated whether serum addition had a positive effect on cell proliferation and viability in either gel configuration. The gels were stored for four weeks in a medium supplemented or not with 20% (v/v foetal calf serum. Throughout a culture period of four weeks, cell proliferation and cell viability were assayed by optical microscopy after provision of Trypan Blue. Non-elaborate culture conditions (room temperature, non-CO2 enriched culture atmosphere were applied throughout the experimental period in order to evaluate cell viability under less than optimal storage conditions. Immobilization of electroporated cells was associated with an initially reduced cell viability, which was gradually increased. Immobilization was associated with maintenance of cell growth for the duration of the experimental period, whereas electroporated cells essentially died after a week in suspension culture. Considerable proliferation of immobilized cells was observed in spherical alginate beads. In both gel configurations, addition of serum was associated with increased cell proliferation. The results of the present study could contribute to an improvement of the storability of biosensors based on electroporated, genetically or membrane-engineered cells.

  14. Surface composition gradients of immobilized cell signaling molecules. Epidermal growth factor on gold

    Energy Technology Data Exchange (ETDEWEB)

    Wang Qian [Department of Chemistry and Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL 61801 (United States); Bohn, Paul W. [Department of Chemistry and Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL 61801 (United States)]. E-mail: bohn@scs.uiuc.edu

    2006-08-14

    Surface composition gradients of the signaling molecule, epidermal growth factor (EGF), have been prepared by an adaptation of the electrochemical gradient technique. EGF is covalently bound to the reactive component, 11-amino-l-undecanethiol (AUT), in a counterpropagating two-component gradient composed of AUT and poly(ethylene glycol) thiol (PEG) using carbodiimide coupling chemistry. Areas of the surface presenting -NH{sub 2} termination react with succinimidyl esters of solvent-accessible acidic amino acids in EGF, while non-specific protein adsorption is resisted in the PEG regions. The maximum surface coverage of EGF prepared in this manner was determined by surface plasmon resonance reflectometry (SPR) on spatially uniform films to be 20% < {gamma} {sub EGF} < 70% depending on the concentration of the EGF derivatization solution. EGF retains its biological activity with this immobilization process, as verified by culturing human umbilical vein endothelial cell (HUVEC) on an EGF-terminated surface for 24 h. PEG shows good resistance to EGF physical adsorption as demonstrated by both SPR and X-ray photoelectron spectroscopy (XPS). The N / C ratio of EGF gradients, which is characteristic of EGF adsorption, because only the protein contains N, while both protein and PEG contain C, was spatially mapped with XPS. The gradient composition distributions are sigmoidal with lateral distance, with the position of the gradient transition region being readily controlled by adjusting the applied potential window. EGF gradients with variable quantitative surface coverage profiles were generated by varying EGF and AUT concentrations.

  15. A new technological approach proposed for distillate production using immobilized cells.

    Science.gov (United States)

    Loukatos, Paul; Kanellaki, Maria; Komaitis, Michael; Athanasiadis, Ilias; Koutinas, Athanasios A

    2003-01-01

    A new technological approach to distillate production using immobilized cells was investigated. The effect of temperature on the main volatile by-products in distillates was determined. Wines produced by delignified cellulose-, gluten- and kissiris-supported biocatalysis were used as starting materials. The produced distillates were analyzed for ethanol, methanol, acetaldehyde, ethyl acetate, propanol-1, isobutanol and amyl alcohol content. The results showed that distillates from delignified cellulosic material (DCM) at 16 degrees C contained smaller amounts of amyl alcohols, 57% of that produced by gluten and 32% of that produced by kissiris. The ethyl acetate content of distillates from DCM improved the aroma of distillates. These results agree with those of sensory evaluation. Subsequently, the scale-up for low-temperature distillate production at 16 degrees C using DCM was further investigated. A new version of an industrial multi-stage fixed bed tower (MFBT) bioreactor with a capacity of 11,000 l proved to be suitable for continuous fermentation by DCM-supported biocatalysis. Economic analysis showed a reduction in the cost of almost 30% for distillate production and 78% for wine production.

  16. An ethanol biosensor based on a bacterial cell-immobilized eggshell membrane

    Institute of Scientific and Technical Information of China (English)

    Guang Ming Wen; Shao Min Shuang; Chuan Dong; Martin M.F. Choi

    2012-01-01

    An ethanol biosensor was fabricated based on a Methylobacterium organophilium-immobilized eggshell membrane and an oxygen (O2) electrode.A linear response for ethanol was obtained in the range of 0.050-7.5 mmol/L with a detection limit of 0.025 mmol/L (S/N =3) and a R.S.D.of 2.1%.The response time was less than 100 s at room temperature and ambient pressure.The optimal loading of bacterial cells on the biosensor membrane is 40 mg (wet weight).The optimal working conditions for the microbial biosensor are pH 7.0 phosphate buffer (50 mmol/L) at 20-25 ℃.The interference test,operational and storage stability of the biosensor are studied in detail.Finally,the biosensor is applied to determine the ethanol contents in various alcohol samples and the results are comparable to that obtained by gas chromatographic method and the results are satisfactory.Our proposed biosensor provides a convenient,simple and reliable method to determine ethanol content in alcoholic drinks.

  17. New biosensor for detection of copper ions in water based on immobilized genetically modified yeast cells.

    Science.gov (United States)

    Vopálenská, Irena; Váchová, Libuše; Palková, Zdena

    2015-10-15

    Contamination of water by heavy metals represents a potential risk for both aquatic and terrestrial organisms, including humans. Heavy metals in water resources can come from various industrial activities, and drinking water can be ex-post contaminated by heavy metals such as Cu(2+) from house fittings (e.g., water reservoirs) and pipes. Here, we present a new copper biosensor capable of detecting copper ions at concentrations of 1-100 μM. This biosensor is based on cells of a specifically modified Saccharomyces cerevisiae strain immobilized in alginate beads. Depending on the concentration of copper, the biosensor beads change color from white, when copper is present in concentrations below the detection limit, to pink or red based on the increase in copper concentration. The biosensor was successfully tested in the determination of copper concentrations in real samples of water contaminated with copper ions. In contrast to analytical methods or other biosensors based on fluorescent proteins, the newly designed biosensor does not require specific equipment and allows the quick detection of copper in many parallel samples.

  18. Time-Course Changes of Steroidogenic Gene Expression and Steroidogenesis of Rat Leydig Cells after Acute Immobilization Stress

    Directory of Open Access Journals (Sweden)

    Han Lin

    2014-11-01

    Full Text Available Leydig cells secrete testosterone, which is essential for male fertility and reproductive health. Stress increases the secretion of glucocorticoid (corticosterone, CORT; in rats, which decreases circulating testosterone levels in part through a direct action by binding to the glucocorticoid receptors (NR3C1 in Leydig cells. The intratesticular CORT level is dependent on oxidative inactivation of glucocorticoid by 11β-hydroxysteroid dehydrogenase 1 (HSD11B1 in Leydig cells. In the present study, we investigated the time-course changes of steroidogenic gene expression levels after acute immobilization stress in rats. The plasma CORT levels were significantly increased 0.5, 1, 3 and 6 h after immobilization stress, while plasma testosterone levels were significantly reduced 3 and 6 h, after stress and luteinizing hormone (LH did not change. Immobilization stress caused the down-regulation of Scarb1, Star and Cyp17a1 expression levels in the rat testis starting at the first hour of stress, ahead of the significant decreases of plasma testosterone levels. Other mRNA levels, including Cyp11a1, Hsd3b1 and Hsd17b3, began to decline after 3 h. Hsd11b1 and Nos2 mRNA levels did not change during the course of stress. Administration of glucocorticoid antagonist RU486 significantly restored plasma testosterone levels. In conclusion, Scarb1, Star and Cyp17a1 expression levels are more sensitive to acute stress, and acute immobilization stress causes the decline of the steroidogenic pathway via elevating the levels of glucocorticoid, which binds to NR3C1 in Leydig cells to inhibit steroidogenic gene expression.

  19. Production of alkaline protease with immobilized cells of bacillus subtilis PE-11 in various matrices by entrapment technique

    OpenAIRE

    Adinarayana, Kunamneni; Jyothi, Bezawada; Ellaiah, Poluri

    2005-01-01

    The purpose of this investigation was to study the effect ofBacillus subtilis PE-11 cells immobilized in various matrices, such as calcium alginate, k-Carrageenan, ployacrylamide, agar-agar, and gelatin, for the production of alkaline protease. Calcium alginate was found to be an effective and suitable matrix for higher alkaline protease productivity compared to the other matrices studied. All the matrices were selected for repeated batch fermentation. The average specific volumetric producti...

  20. Effect of concentration and substrate flow rate on isomaltulose production from sucrose by Erwinia sp. cells immobilized in calcium-alginate using packed bed reactor.

    Science.gov (United States)

    Kawaguti, Haroldo Yukio; Harumi Sato, Hélia

    2010-09-01

    Isomaltulose was obtained from sucrose solution by immobilized cells of Erwinia sp. D12 using a batch and a continuous process. Parameters for sucrose conversion into isomaltulose were evaluated using both experimental design and response surface methodology. Erwinia sp. D12 cells were immobilized in different alginates, and the influence of substrate flow rate and concentration parameters to produce isomaltulose from sucrose were observed. Response surface methodology demonstrated that packed bed columns containing cells immobilized in low-viscosity sodium alginate (250 cP) presented a mean isomaltulose conversion rate of 47%. In a continuous process, both sucrose substrate concentration and substrate flow rate parameters had a significant effect (p < 0.05) and influenced the conversion of sucrose into isomaltulose. Higher conversion rates of sucrose into isomaltulose, from 53-75% were obtained using 75 g of immobilized cells at a substrate flow rate of 0.6 mL/min.

  1. Use of an Immobilized Monoclonal Antibody to Examine Integrin &agr;5&bgr;1 Signaling Independent of Cell Spreading

    Directory of Open Access Journals (Sweden)

    Bao Wenjie

    2002-01-01

    Full Text Available Cell attachment to the extracellular matrix (ECM engages integrin signaling into the cell, but part of the signaling response also stem from cell spreading (3. To analyze specific integrin signaling-mediated responses independent of cell spreading, we developed a method engaging integrin signaling by use of an immobilized anti-integrin monoclonal antibody (mab directed against the fibronectin (FN receptor integrin &agr;5&bgr;1. ECV 304 cells were plated onto FN or immobilized mab JBS5 (anti-integrin &agr;5&bgr;1 or onto poly-L-lysin (P-L-L, which mediates integrin-independent attachment. Cells attached and spread on FN, while cells on JBS5 or P-L-L attached but did not spread. Importantly, plating onto FN or mab JBS5 gave rise to identical integrin-induced responses, including a down-regulation of the cyclin-dependent kinase (Cdk2 inhibitors p21CIP1 and p27KIP1, while attachment to P-L-L did not. We conclude that engagement of the FN-receptor integrin &agr;5&bgr;1 induces integrin signaling regulating the Cdk2-inhibitors independent of cell spreading and present a method for how integrin signaling can be analyzed separate from the effects of cell spreading.

  2. Detection of Sperm DNA Damage in Workers Exposed to Benzene by Modified Single Cell Gel Electrophoresis

    Institute of Scientific and Technical Information of China (English)

    Bo SONG; Zhi-ming CAI; Xin LI; Li-xia DENG; Qiao ZHANG; Lu-kang ZHENG

    2005-01-01

    Objective To assess the effect of benzene on sperm DNA damageMethods Twenty-seven benzene-exposed workers were selected as exposed groupand 35 normal sperm donors as control group. Air concentration of benzene series inworkshop was determined by gas chromatography. As an internal exposure dose ofbenzene, the concentration of trans, trans-muconic acid (ttMA) was determined byhigh performance liquid chromatography. DNA was detected by modified single cellgel electrophoresis (SCGE).Results The air concentrations of benzene, toluene and xylene at the workplace were86.49 ± 2.83 mg/m3, 97.20 ±3.52 mg/m3 and 97.45 ±2.10 mg/m3, respectively.Urinary ttMA in exposed group (1.040 ± 0.617 mg/L) was significantly higher thanthat of control group (0.819 ± 0.157 mg/L). The percentage of head DNA, determinedby modified SCGE method, significantly decreased in the exposed group (n=13, 70.18%± 7.36%) compared with the control (n=16, 90.62% ± 2.94%)(P<0.001).Conclusion The modified SCGE method can be used to investigate the damage ofsperm DNA. As genotoxin and reprotoxins, benzene had direct effect on the germ cellsduring the spermatogenesiss.

  3. Interaction between immobilized polyelectrolyte complex nanoparticles and human mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Woltmann B

    2014-05-01

    Full Text Available Beatrice Woltmann,1 Bernhard Torger,2,3 Martin Müller,2,3,* Ute Hempel1,*1Dresden University of Technology, Faculty of Medicine Carl Gustav Carus, Institute of Physiological Chemistry, Dresden, Germany; 2Leibniz Institute of Polymer Research Dresden, Department of Polyelectrolytes and Dispersions, Dresden, Germany; 3Dresden University of Technology, Department of Chemistry and Food Chemistry, Dresden, Germany*These authors contributed equally to this workBackground: Implant loosening or deficient osseointegration is a major problem in patients with systemic bone diseases (eg, osteoporosis. For this reason, the stimulation of the regional cell population by local and sustained drug delivery at the bone/implant interface to induce the formation of a mechanical stable bone is promising. The purpose of this study was to investigate the interaction of polymer-based nanoparticles with human bone marrow-derived cells, considering nanoparticles’ composition and surface net charge.Materials and methods: Polyelectrolyte complex nanoparticles (PECNPs composed of the polycations poly(ethyleneimine (PEI, poly(L-lysine (PLL, or (N,N-diethylaminoethyldextran (DEAE in combination with the polyanions dextran sulfate (DS or cellulose sulfate (CS were prepared. PECNPs’ physicochemical properties (size, net charge were characterized by dynamic light scattering and particle charge detector measurements. Biocompatibility was investigated using human mesenchymal stromal cells (hMSCs cultured on immobilized PECNP films (5–50 nmol·cm-2 by analysis for metabolic activity of hMSCs in dependence of PECNP surface concentration by MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt assay, as well as cell morphology (phase contrast microscopy.Results: PECNPs ranging between ~50 nm and 150 nm were prepared. By varying the ratio of polycations and polyanions, PECNPs with a slightly positive (PEC+NP or negative (PEC

  4. Immobilization of Lactobacillus rhamnosus in mesoporous silica-based material: An efficiency continuous cell-recycle fermentation system for lactic acid production.

    Science.gov (United States)

    Zhao, Zijian; Xie, Xiaona; Wang, Zhi; Tao, Yanchun; Niu, Xuedun; Huang, Xuri; Liu, Li; Li, Zhengqiang

    2016-06-01

    Lactic acid bacteria immobilization methods have been widely used for lactic acid production. Until now, the most common immobilization matrix used is calcium alginate. However, Ca-alginate gel disintegrated during lactic acid fermentation. To overcome this deficiency, we developed an immobilization method in which Lactobacillus rhamnosus cells were successfully encapsulated into an ordered mesoporous silica-based material under mild conditions with a high immobilization efficiency of 78.77% by using elemental analysis. We also optimized the cultivation conditions of the immobilized L. rhamnosus and obtained a high glucose conversion yield of 92.4%. Furthermore, L. rhamnosus encapsulated in mesoporous silica-based material exhibited operational stability during repeated fermentation processes and no decrease in lactic acid production up to 8 repeated batches.

  5. Implementation of Transfer Evolution Formalism (TEF) to the modelization of strongly coupled thermodynamic systems: thermocapillar pumping fluid loop, Peltier effect thermoelements, electrophoresis cell

    Energy Technology Data Exchange (ETDEWEB)

    Butto, C.; Fudym, O.; Joly, J.L.; Platel, V.; Zely, D. [Toulouse-3 Univ., 31 (France); Franchisseur, R.; Grandpeix, J.Y.; Lahellec, A. [Centre National de la Recherche Scientifique (CNRS), 91 - Orsay (France)

    1993-12-31

    This text presents the interest of TEF (Transfer Evolution Formalism) method in three cases: thermocapillar pumping, Peltier effect thermoelements and electrophoresis cell , that illustrate on the one hand, the easiness of modelizing coupled phenomena thanks to TEF, and on the other hand, the use of coupling analysis in order to understand the running of a device. (TEC). figs., refs.

  6. Tris-sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61.

    Science.gov (United States)

    Aydogan, Mehmet Nuri; Taskin, Mesut; Canli, Ozden; Arslan, Nazli Pinar; Ortucu, Serkan

    2014-01-01

    The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris-sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.

  7. The Cutting Edge of Affinity Electrophoresis Technology

    Directory of Open Access Journals (Sweden)

    Eiji Kinoshita

    2015-03-01

    Full Text Available Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years.

  8. The Cutting Edge of Affinity Electrophoresis Technology

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years.

  9. Selected complete blood cell count and plasma protein electrophoresis parameters in pet psittacine birds evaluated for illness.

    Science.gov (United States)

    Briscoe, Jeleen A; Rosenthal, Karen L; Shofer, Frances S

    2010-06-01

    Veterinarians rely on results of both the complete blood cell count (CBC) and plasma protein electrophoresis (EPH) in conjunction with the results of the plasma biochemical analysis to evaluate the health status of avian patients. Because the CBC and protein EPH measure different aspects of the immune response to disease, both tests are recommended in avian patients to rule out infectious or inflammatory disease. To evaluate results of the CBC and protein EPH in pet psittacine birds, the records of 144 pet psittacine birds, comprising 11 genera, that were presented for suspected illness were reviewed. Results of the CBC (total white blood cell count and packed cell volume) and protein EPH (alpha, beta, and gamma globulin concentrations) from submitted blood samples from each bird were evaluated. Of the 144 birds, 63 (43.8%) had abnormal CBC results, and 25 (17.4%) had abnormal EPH measurements. Results of the CBC and protein EPH were within reference ranges in 73 birds (50.7%). Abnormal results of the CBC in conjunction with normal EPH results were present in 46 birds (31.9%), compared with 8 birds (5.6%) with normal results of the CBC and abnormal EPH results. The findings of this study could aid practitioners in evaluating psittacine patients and prioritizing the value of individual diagnostic tests.

  10. Electrophoresis experiments in microgravity

    Science.gov (United States)

    Snyder, Robert S.; Rhodes, Percy H.

    1991-01-01

    The use of the microgravity environment to separate and purify biological cells and proteins has been a major activity since the beginning of the NASA Microgravity Science and Applications program. Purified populations of cells are needed for research, transplantation and analysis of specific cell constituents. Protein purification is a necessary step in research areas such as genetic engineering where the new protein has to be separated from the variety of other proteins synthesized from the microorganism. Sufficient data are available from the results of past electrophoresis experiments in space to show that these experiments were designed with incomplete knowledge of the fluid dynamics of the process including electrohydrodynamics. However, electrophoresis is still an important separation tool in the laboratory and thermal convection does limit its performance. Thus, there is a justification for electrophoresis but the emphasis of future space experiments must be directed toward basic research with model experiments to understand the microgravity environment and fluid analysis to test the basic principles of the process.

  11. Optimizing immobilized enzyme performance in cell-free environments to produce liquid fuels.

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Sanat

    2015-02-05

    The overall goal of this project was to optimize enzyme performance for the production of bio-diesel fuel. Enzyme immobilization has attracted much attention as a means to increase productivity. Mesorporous silica materials have been known to be best suited for immobilizing enzymes. A major challenge is to ensure that the enzymatic activity is retained after immobilization. Two major factors which drive enzymatic deactivation are protein-surface and inter-protein interactions. Previously, we studied protein stability inside pores and how to optimize protein-surface interactions to minimize protein denaturation. In this work we studied eh effect of surface curvature and chemistry on inter-protein interactions. Our goal was to find suitable immobilization supports which minimize these inter-protein interactions. Our studies carried out in the frame work of Hydrophobic-Polar (HP) model showed that enzymes immobilized inside hydrophobic pores of optimal sizes are best suited to minimize these inter-protein interactions. Besides, this study is also of biological importance to understand the role of chaperonins in protein disaggregation. Both of these aspects profited immensely with collaborations with our experimental colleague, Prof. Georges Belfort (RPI), who performed the experimental analog of our theoretical works.

  12. Quantitation of residual trypsin in cell-based therapeutics using immobilized α-1-antitrypsin or SBTI in an ELISA format.

    Science.gov (United States)

    Braatz, James A; Elias, Christopher; Finny, Joseph G; Tran, Huan; McCaman, Michael

    2015-02-01

    An Enzyme-Linked Immunosorbent Assay (ELISA) has been developed for the quantitation of porcine trypsin as a process residual in cell therapy products based on its capture by either of two immobilized anti-trypsins, α-1-antitrypsin (α1AT) or soybean trypsin inhibitor (SBTI) followed by detection with a polyclonal goat anti-porcine trypsin-IgG conjugated with peroxidase. It was demonstrated that an extended range of antigen quantitation could be achieved that covered nearly three orders of magnitude of trypsin concentration. The utility of the assay was demonstrated by its application to samples generated in a cell-based therapeutic manufacturing setting.

  13. Soymilk residue (okara as a natural immobilization carrier for Lactobacillus plantarum cells enhances soymilk fermentation, glucosidic isoflavone bioconversion, and cell survival under simulated gastric and intestinal conditions

    Directory of Open Access Journals (Sweden)

    Xia Xiudong

    2016-11-01

    Full Text Available Cell immobilization is an alternative to microencapsulation for the maintenance of cells in a liquid medium. However, artificial immobilization carriers are expensive and pose a high safety risk. Okara, a food-grade byproduct from soymilk production, is rich in prebiotics. Lactobacilli could provide health enhancing effects to the host. This study aimed to evaluate the potential of okara as a natural immobilizer for L. plantarum 70810 cells. The study also aimed to evaluate the effects of okara-immobilized L. plantarum 70810 cells (IL on soymilk fermentation, glucosidic isoflavone bioconversion, and cell resistance to simulated gastric and intestinal stresses. Scanning electron microscopy (SEM was used to show cells adherence to the surface of okara. Lactic acid, acetic acid and isoflavone analyses in unfermented and fermented soymilk were performed by HPLC with UV detection. Viability and growth kinetics of immobilized and free L. plantarum 70810 cells (FL were followed during soymilk fermentation. Moreover, changes in pH, titrable acidity and viscosity were measured by conventional methods. For in vitro testing of simulated gastrointestinal resistance, fermented soymilk was inoculated with FL or IL and an aliquot incubated into acidic MRS broth which was conveniently prepared to simulate gastric, pancreatic juices and bile salts. Survival to simulated gastric and intestinal stresses was evaluated by plate count of colony forming units on MRS agar. SEM revealed that the lactobacilli cells attached and bound to the surface of okara. Compared with FL, IL exhibited a significantly higher specific growth rate, shorter lag phase of growth, higher productions of lactic and acetic acids, a faster decrease in pH and increase in titrable acidity, and a higher soymilk viscosity. Similarly, IL in soymilk showed higher productions of daizein and genistein compared with the control. Compared with FL, IL showed reinforced resistance to simulatedgastric and

  14. Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence.

    Science.gov (United States)

    de Campos, Richard P S; Siegel, Joseph M; Fresta, Claudia G; Caruso, Giuseppe; da Silva, José A F; Lunte, Susan M

    2015-09-01

    Superoxide, a naturally produced reactive oxygen species (ROS) in the human body, is involved in many pathological and physiological signaling processes. However, if superoxide formation is left unregulated, overproduction can lead to oxidative damage to important biomolecules, such as DNA, lipids, and proteins. Superoxide can also lead to the formation of peroxynitrite, an extremely hazardous substance, through its reaction with endogenously produced nitric oxide. Despite its importance, quantitative information regarding superoxide production is difficult to obtain due to its high reactivity and low concentrations in vivo. MitoHE, a fluorescent probe that specifically reacts with superoxide, was used in conjunction with microchip electrophoresis (ME) and laser-induced fluorescence (LIF) detection to investigate changes in superoxide production by RAW 264.7 macrophage cells following stimulation with phorbol 12-myristate 13-acetate (PMA). Stimulation was performed in the presence and absence of the superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC) and 2-metoxyestradiol (2-ME). The addition of these inhibitors resulted in an increase in the amount of superoxide specific product (2-OH-MitoE(+)) from 0.08 ± 0.01 fmol (0.17 ± 0.03 mM) in native cells to 1.26 ± 0.06 fmol (2.5 ± 0.1 mM) after PMA treatment. This corresponds to an approximately 15-fold increase in intracellular concentration per cell. Furthermore, the addition of 3-morpholino-sydnonimine (SIN-1) to the cells during incubation resulted in the production of 0.061 ± 0.006 fmol (0.12 ± 0.01 mM) of 2-OH-MitoE(+) per cell on average. These results demonstrate that indirect superoxide detection coupled with the use of SOD inhibitors and a separation method is a viable method to discriminate the 2-OH-MitoE(+) signal from possible interferences.

  15. High background radiation areas of Ram sar in Iran: evaluation of DNA damage by alkaline single cell gel electrophoresis (SCE)

    Energy Technology Data Exchange (ETDEWEB)

    Masoomi, J.R. [Biophysics Department, College of Sciences, Tarbiat Modarres University, Tehran (Iran, Islamic Republic of); Mohammadi, Sh. [Radiation Molecular Genetic Laboratory, National Radiation Protection Department (NRPD), Iranian Nuclear Regulatory Authority (INRA), P.O. Box 14155-4494, Tehran (Iran, Islamic Republic of); Amini, M. [Faculty of Pharmacy, Azad University of Tehran (Iran, Islamic Republic of); Ghiassi-Nejad, M. [Biophysics Department, College of Sciences, Tarbiat Modarres University, Tehran (Iran, Islamic Republic of)]. E-mail: ghiassi@mailcity.com

    2006-07-01

    The hot springs in special areas in Ram sar, a northern coastal town in Iran, contain {sup 226}Ra and {sup 222}Rn. The natural radiation effects, radiosensitivity or adaptive responses, on the inhabitants of high natural radiation in Ram sar were studied. The single cell gel electrophoresis was used to monitor DNA damages. Three groups of volunteers were selected, one from high natural background radiation areas as the case group and two from normal background radiation areas as controls (control 1 and control 2). The latter one had the similar living situation to case group while the other (control 2) had different living situation from the other groups. Peripheral blood mononuclear cells (PMNCs) were separated and irradiated by {sup 6}Co source at five different gamma doses. It was found that the spontaneous level of DNA damage and the induced DNA damage in all challenging doses in case group was considerably higher than control groups (p < 0.05). On the other hand, the repair rate in those volunteers, who received less than 10.2 mSv/y was significantly more than the control groups. In the contrary, individuals who live in homes with more than 10.2 mSv/y had incomplete repair. Additionally the plasma and urinary levels of vitamin C were measured spectrophotometrically. Although the concentration of vitamin C of plasma was equal in case and control 1 groups, the urinary level of vitamin C was found to be lower in the case group.

  16. Hemicellulosic Ethanol Production by Immobilized Wild Brazilian Yeast Scheffersomyces shehatae UFMG-HM 52.2: Effects of Cell Concentration and Stirring Rate.

    Science.gov (United States)

    Antunes, F A F; Santos, J C; Chandel, A K; Milessi, T S S; Peres, G F D; da Silva, S S

    2016-02-01

    The use of sugarcane bagasse hemicellulosic hydrolysates presents an interesting alternative to second generation (2G) ethanol production. Techniques to enhance the fermentation process, e.g., the use of immobilized cells, is one of the key factors for efficient production. Here, the effect of two important parameters (cell concentration in immobilized system and stirring rate) on the 2G ethanol production using the wild Brazilian yeast S. shehatae UFMG-HM 52.2 immobilized in calcium alginate matrix are presented. A 2(2) full factorial design of experiments was carried out to evaluate the effect of cell concentrations in sodium alginate solution for immobilized bead production (3.0, 6.0, and 9.0 g/L) and stirring rate (150, 200, and 250 rpm) for 2G ethanol production. Statistical analysis showed that the use of both variables at low levels enhanced ethanol yield (YP/S). Under these process conditions, YP/S of 0.31 g/g and ethanol productivity (Qp) of 0.12 g/L h were achieved. Results showed the potential of this immobilized yeast in 2G ethanol production from C5 sugars and demonstrate the importance of adequate cell concentration in immobilized systems, a finding that stands to increase bioprocesses yields and productivity.

  17. Production of butyric acid from glucose and xylose with immobilized cells of Clostridium tyrobutyricum in a fibrous-bed bioreactor.

    Science.gov (United States)

    Jiang, Ling; Wang, Jufang; Liang, Shizhong; Wang, Xiaoning; Cen, Peilin; Xu, Zhinan

    2010-01-01

    Butyric acid has many applications in chemical, food, and pharmaceutical industries. In the present study, Clostridium tyrobutyricum ATCC 25755 was immobilized in a fibrous-bed bioreactor to evaluate the performance of butyrate production from glucose and xylose. The results showed that the final concentration and yield of butyric acid were 13.70 and 0.46 g g(-1), respectively, in batch fermentation when 30 g L(-1) glucose was introduced into the bioreactor. Furthermore, high concentration 10.10 g L(-1) and yield 0.40 g g(-1) of butyric acid were obtained with 25 g L(-1) xylose as the carbon source. The immobilized cells of C. tyrobutyricum ensured similar productivity and yield from repeated batch fermentation. In the fed-batch fermentation, the final concentration of butyric acid was further improved to 24.88 g L(-1) with one suitable glucose feeding in the fibrous-bed bioreactor. C. tyrobutyricum immobilized in the fibrous-bed bioreactor would provide an economically viable fermentation process to convert the reducing sugars derived from plant biomass into the final bulk chemical (butyric acid).

  18. Use of Pistacia terebinthus resin as immobilization support for Lactobacillus casei cells and application in selected dairy products.

    Science.gov (United States)

    Schoina, Vasiliki; Terpou, Antonia; Angelika-Ioanna, Gialleli; Koutinas, Athanasios; Kanellaki, Maria; Bosnea, Loulouda

    2015-09-01

    Resin from Pistacia terebinthus tree was used for the immobilization of L. casei ATCC 393 cells. The encapsulated L. casei cells biocatalysts were added as adjuncts during yogurt production at 45 °C and probiotic viability was assessed during storage at 4 °C. For comparison reasons yogurt with free L. casei cells were prepared. The effect of encapsulated bacteria as adjuncts in yogurt on pH, lactic acid, lactose and other physicochemical parameters were studied for 60 storage days at 4 °C. Samples were also tested for the microbiological and organoleptic characteristics during storage at 4 °C. Encapsulation matrix seems to sustain the viability of embedded L. casei cells at levels more than 7 logcfug(-1) after 60 days of storage at 4 °C. Furthermore, the absence of pathogens such as Salmonella, Staphylococci, Enterobacteriaceae and coliforms in the produced yogurts is noteworthy where spoilage microorganisms such as yeasts and molds seem to affect yogurt quality only in absence of Pistacia terebinthus resin. The effect of the resin on production of aroma-related compounds responsible for yogurt flavor was also studied using the solid phase microextraction gas chromatography/mass spectrometry technique. Alpha and beta- pinene were the major aroma compounds detected in produced yogurts (over 60 % of total aromatic compounds detected). Yogurts with immobilized cells on P.terebintus resin had a fine aroma and taste characteristic of the resin.

  19. KINETIC STUDIES ON BIODEGRADATION OF LIPIDS FROM OLIVE OIL MILL WASTEWATERS WITH FREE AND IMMOBILIZED Bacillus sp. CELLS

    Directory of Open Access Journals (Sweden)

    Anca-Irina Galaction

    2012-03-01

    Full Text Available The studies on the biodegradation of lipids from olive oil mill wastewater with free and immobilized Bacillus sp. cells indicated that the maximum specific rate of the process is reached at pH = 8. The use of immobilized cells allows to increasing the number of biodegradation process cycles, but reduces the rate of the process. In this case, the process rate depends on the biocatalysts size and cells concentration inside them. Thus, at bacterial cells concentration of 9 g d.w./100 mL biocatalyst, the apparent specific rate varied from 4.65 to 1.46×10-2 h-1 by increasing the biocatalyst particles diameter from 3 to 4.2 mm.The cumulated influences of the particles size and cells concentration have been included in a mathematical model for the apparent specific rate of lipids biodegradation. The model offers a good concordance with the experimental data, the average deviation being of +/- 7.38%.

  20. Enhanced production of alkaline thermostable keratinolytic protease from calcium alginate immobilized cells of thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity.

    Science.gov (United States)

    Shrinivas, Dengeti; Kumar, Raghwendra; Naik, G R

    2012-01-01

    The thermoalkalophilic Bacillus halodurans JB 99 cells known for production of novel thermostable alkaline keratinolytic protease were immobilized in calcium alginate matrix. Batch and repeated batch cultivation using calcium alginate immobilized cells were studied for alkaline protease production in submerged fermentation. Immobilized cells with 2.5% alginate and 350 beads/flask of initial cell loading showed enhanced production of alkaline protease by 23.2% (5,275 ± 39.4 U/ml) as compared to free cells (4,280 ± 35.4 U/ml) after 24 h. In the semicontinuous mode of cultivation, immobilized cells under optimized conditions produced an appreciable level of alkaline protease in up to nine cycles and reached a maximal value of 5,975 U/ml after the seventh cycle. The enzyme produced from immobilized cells efficiently degraded chicken feathers in the presence of a reducing agent which can help the poultry industry in the management of keratin-rich waste and obtaining value-added products.

  1. The effect of VEGF-immobilized nickel-free high-nitrogen stainless steel on viability and proliferation of vascular endothelial cells.

    Science.gov (United States)

    Sasaki, Makoto; Inoue, Motoki; Katada, Yasuyuki; Taguchi, Tetsushi

    2012-04-01

    Using ester bonds, vascular endothelial growth factor-A (VEGF-A) was immobilized on the surface of a novel biometal, nickel-free high-nitrogen stainless steel (HNS). The biological activity of immobilized VEGF-A was investigated after the culture of human umbilical vein endothelial cells (HUVECs) on the substrate. Immobilization of VEGF-A onto the HNS surface was performed using trisuccinimidyl citrate (TSC) as a linker. Firstly, UV irradiation was employed to amplify hydroxyl groups on the HNS surface. Next, the HNS was dipped into TSC/dimethyl sulfoxide solution at room temperature. From the results of water contact angle measurement and X-ray photoelectron spectroscopy (XPS) analysis, TSC was found to be immobilized on the HNS surface via ester bonds. Quantitative analysis demonstrated that immobilized VEGF-A remained even after immersion in culture medium for 7 days; however, it was gradually deimmobilized by hydrolysis of the ester bonds at the TSC-metal interface. As a result, VEGF-A-immobilized HNS significantly contributed to the stimulation of HUVEC growth for the initial stage of culture, even though the gradual reduction in growth stimulation of HUVECs occurred by the sequential deimmobilization of VEGF-A, which was caused by the hydrolysis of the ester groups. Therefore, VEGF-A-immobilized HNS could be applied as a basic material for coronary stents.

  2. 海澡酸钙胶囊化重组E.coli BL21(DE3)生产靛蓝%Production of Indigo by Immobilization of E.coli BL21 (DE3) Cells in Calcium- Alginate Gel Capsules

    Institute of Scientific and Technical Information of China (English)

    陆燕; 梅乐和

    2007-01-01

    The ability of catalyzing indole into indigo of gene engineering strain expressing P450 BM3 immobilized by entrapment in calcium-alginate gel capsules was examined, and various characteristics of immobilized cells were assessed.Optimum conditions for cells activity were not affected after immobilization, and pH and temperature for both free and immobilized cells were found to be pH 7.5 and 35℃, respectively.The immobilized cells exhibited a markedly improved thermal stability than free cells.After five repeated experiments, the yield of indigo with the immobilized cells retained over 94% of their original activity, which indicated that the operational stability for recycling in batch processes was improved.

  3. Comparison of Yeast Cell Protein Solubilization Procedures for Two-dimensional Electrophoresis

    DEFF Research Database (Denmark)

    Harder, A; Wildgruber, R; Nawrocki, A;

    1999-01-01

    Three different procedures for the solubilization of yeast (S. cerevisiae) cell proteins were compared on the basis of the obtained two-dimensional (2-D) polypeptide patterns. Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high ...

  4. Aerobic decolorization and degradation of Acid Orange G (AOG) by suspended growing cells and immobilized cells of a yeast strain Candida tropicalis TL-F1.

    Science.gov (United States)

    Tan, Liang; Li, Hua; Ning, Shuxiang; Hao, Jia

    2014-10-01

    In this study, aerobic decolorization and degradation of azo dye Acid Orange G (AOG) by both suspended growing cells and immobilized cells of a yeast strain Candida tropicalis TL-F1 were studied. The effects of different parameters on decolorization of AOG by both growing suspended and immobilized strain TL-F1 were investigated. Furthermore, a possible decolorization mechanism of AOG was proposed through analyzing metabolic intermediates using UV-vis and high-performance liquid chromatography-mass spectrometry (HPLC-MS) methods. Strain TL-F1 could decolorize AOG in both liquid and solid mediums through degradation. The optimal conditions for decolorization with suspended growing cells of strain TL-F1 were as follows: 6-10 g/L sucrose, 5-7 g/L urea, ≥6 % (v/v) inoculation size, ≥160 rpm, 35-40 °C, and pH 5.0-6.0; and those for immobilized cells, the conditions were as follows: 4-6 g/L glucose, 0.2-0.4 g/L urea, 6-10 g/L (wet cell pellets) inoculation size, ≥160 rpm, 35-40 °C, and pH 5.0-7.0. Results of UV-vis scanning spectra suggested that AOG was decolorized through biodegradation, and the possible pathway was proposed through the results of HPLC-MS analysis and related literature. This is a systematic research on aerobic decolorization and degradation of AOG by both suspended and immobilized cells of a C. tropicalis strain.

  5. Analysis of differentially expressed mitochondrial proteins in chromophobe renal cell carcinomas and renal oncocytomas by 2-D gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Maria V. Yusenko, Thomas Ruppert, Gyula Kovacs

    2010-01-01

    Full Text Available Renal oncocytomas (RO and chromophobe renal cell carcinomas (RCC display morphological and functional alterations of the mitochondria. Previous studies showed that accumulation of mitochondria in ROs is associated with somatic mutations of mitochondrial DNA (mtDNA resulting in decreased activity of the respiratory chain complex I, whereas in chromophobe RCC only heteroplasmic mtDNA mutations were found. To identify proteins associated with these changes, for the first time we have compared the mitochondrial proteomes of mitochondria isolated from ROs and chromophobe RCCs as well as from normal kidney tissues by two-dimensional polyacrylamide gel electrophoresis. The proteome profiles were reproducible within the same group of tissues in subsequent experiments. The expression patterns within each group of samples were compared and 81 in-gel digested spots were subjected to nanoLC-MS/MS-based identification of proteins. Although the list of mitochondrial proteins identified in this study is incomplete, we identified the downregulation of NDUFS3 from complex I of the respiratory chain and upregulation of COX5A, COX5B, and ATP5H from complex IV and V in ROs. In chromophobe RCCs downregulation of ATP5A1, the alpha subunit of complex V, has been observed, but no changes in expression of other complexes of the respiratory chain were detected. To confirm the role of respiratory chain complex alterations in the morphological and/or functional changes in chromophobe RCCs and ROs, further studies will be necessary.

  6. Avoiding acidic region streaking in two-dimensional gel electrophoresis: Case study with two bacterial whole cell protein extracts

    Indian Academy of Sciences (India)

    Arnab Roy; Umesh Varshney; Debnath Pal

    2014-09-01

    Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.

  7. Bioethanol production from starchy biomass by direct fermentation using saccharomyces diastaticus in batch free and immobilized cell systems

    Energy Technology Data Exchange (ETDEWEB)

    Kilonzo, P.M.; Margaritis, A. [University of Western Ontario, London, ON (Canada). Dept. of Chemical and Biochemical Engineering; Yu, J.; Ye, Q. [East China Univ. of Science and Technology, Shanghai (China). Biochemical Engineering Research Inst. and State Key Lab

    2006-07-01

    The feasibility of using amylolytic yeasts for the direct fermentation of starchy biomass to ethanol was discussed. Although amylolytic yeasts such as Saccharomycopsis, Lipomyces, and Schwaniomyces secrete both {alpha}-amylase and glucoamylase enzymes that synergistically enhance starch degradation, they are not suitable for industrial bio-ethanol production because of low tolerance for ethanol and slow fermentation rate. For that reason, this study examined the direct ethanol fermentation of soluble starch or dextrin with the amylolytic yeast Saccharomyces diastaticus in batch free and immobilized cells systems. Saccharomyces diastaticus secretes glucoamylase and can therefore assimilate and ferment starch and starch-like biomass. The main focus of the study was on parameters leading to higher ethanol yields from high concentration of dextrin and soluble starch using batch cultures. A natural attachment method was proposed in which polyurethane foam sheets were used as the carrier for amylolytic yeasts immobilization in ethanol fermentations. The support was chosen because it was inexpensive, autoclavable, pliable and could be tailored to suit process requirements regarding net surface charge, shape and size. It was found that Saccharomyces diastaticus was very efficient in terms of fermentation of high initial concentrations of dextrin or soluble starch. Higher concentrations of ethanol were produced. In batch fermentations, the cells fermented high dextrin concentrations more efficiently. In particular, in batch fermentation, more than 92 g-L of ethanol was produced from 240 g-L of dextrin, at conversion efficiency of 90 per cent. The conversion efficiency decreased to 60 per cent but a higher final ethanol concentration of 147 g/L was attained with a medium containing 500 g/L of dextrin. In an immobilized cell bioreactor, Saccharomyces diastaticus produced 83 g/L of ethanol from 240 g/L of dextrin, corresponding to ethanol volumetric productivity of 9.1 g

  8. Biodegradation of benzidine based azodyes Direct red and Direct blue by the immobilized cells of Pseudomonas fluorescens D41.

    Science.gov (United States)

    Puvaneswari, N; Muthukrishnan, J; Gunasekaran, P

    2002-10-01

    Benzidine based azodyes are proven carcinogens, mutagens and have been linked to bladder cancer of human beings and laboratory animals. The textile and dyestuff manufacturing industry are the two major sources that released azodyes in their effluents. The dye, Direct blue contains two carcinogenic compounds namely benzidine (BZ), 4-amino biphenyl (4-ABP), while the dye Direct red has benzidine (BZ). Among 40 isolates of Pseudomonas fluorescens screened, one isolate designated as D41 was found to be capable of extensively degrading the dyes Direct blue and Direct red. Immobilized cells of P. fluorescens D41 efficiently degraded Direct red (82%) and Direct blue (71%) in the presence of glucose.

  9. Surface modification of silicone tubes by functional carboxyl and amine, but not peroxide groups followed by collagen immobilization improves endothelial cell stability and functionality.

    Science.gov (United States)

    Salehi-Nik, Nasim; Amoabediny, Ghassem; Shokrgozar, Mohammad Ali; Mottaghy, Khosrow; Klein-Nulend, Jenneke; Zandieh-Doulabi, Behrouz

    2015-03-02

    Surface modification by functional groups promotes endothelialization in biohybrid artificial lungs, but whether it affects endothelial cell stability under fluid shear stress, and the release of anti-thrombotic factors, e.g. nitric oxide (NO), is unknown. We aimed to test whether surface-modified silicone tubes containing different functional groups, but similar wettability, improve collagen immobilization, endothelialization, cell stability and cell-mediated NO-release. Peroxide, carboxyl, and amine-groups increased collagen immobilization (41-76%). Only amine-groups increased ultimate tensile strength (2-fold). Peroxide and amine enhanced (1.5-2.5 fold), but carboxyl-groups decreased (2.9-fold) endothelial cell number after 6 d. After collagen immobilization, cell numbers were enhanced by all group-modifications (2.8-3.8 fold). Cells were stable under 1 h-fluid shear stress on amine, but not carboxyl or peroxide-group-modified silicone (>50% cell detachment), while cells were also stable on carboxyl-group-modified silicone with immobilized collagen. NO-release was increased by peroxide and amine (1.1-1.7 fold), but decreased by carboxyl-group-modification (9.8-fold), while it increased by all group-modifications after collagen immobilization (1.8-2.8 fold). Only the amine-group-modification changed silicone stiffness and transparency. In conclusion, silicone-surface modification of blood-contacting parts of artificial lungs with carboxyl and amine, but not peroxide-groups followed by collagen immobilization allows the formation of a stable functional endothelial cell layer. Amine-group-modification seems undesirable since it affected silicone's physical properties.

  10. Antimicrobial and cell viability measurement of bovine serum albumin capped silver nanoparticles (Ag/BSA) loaded collagen immobilized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film.

    Science.gov (United States)

    Bakare, Rotimi; Hawthrone, Samantha; Vails, Carmen; Gugssa, Ayele; Karim, Alamgir; Stubbs, John; Raghavan, Dharmaraj

    2016-03-01

    Bacterial infection of orthopedic devices has been a major concern in joint replacement procedures. Therefore, this study is aimed at formulating collagen immobilized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film loaded with bovine serum albumin capped silver nanoparticles (Ag/BSA NPs) to inhibit bacterial growth while retaining/promoting osteoblast cells viability. The nanoparticles loaded collagen immobilized PHBV film was characterized for its composition by X-ray Photoelectron Spectroscopy and Anodic Stripping Voltammetry. The extent of loading of Ag/BSA NPs on collagen immobilized PHBV film was found to depend on the chemistry of the functionalized PHBV film and the concentration of Ag/BSA NPs solution used for loading nanoparticles. Our results showed that more Ag/BSA NPs were loaded on higher molecular weight collagen immobilized PHEMA-g-PHBV film. Maximum loading of Ag/BSA NPs on collagen immobilized PHBV film was observed when 16ppm solution was used for adsorption studies. Colony forming unit and optical density measurements showed broad antimicrobial activity towards Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa at significantly lower concentration i.e., 0.19 and 0.31μg/disc, compared to gentamicin and sulfamethoxazole trimethoprim while MTT assay showed that released nanoparticles from Ag/BSA NPs loaded collagen immobilized PHBV film has no impact on MCTC3-E1 cells viability.

  11. Kinetics of ethanol production by immobilized Kluyveromyces marxianus cells at varying sugar concentrations of Jerusalem artichoke juice

    Energy Technology Data Exchange (ETDEWEB)

    Bajpai, P.; Margaritis, A.

    1987-08-01

    Kinetics of ethanol fermentation at varying sugar concentrations of Jerusalem artichoke tuber extract has been studied using Kluyveromyces marxianus cells immobilized in calcium alginate gel beads. A maximum ethanol concentration of 111 g/l was achieved at an initial sugar concentration of 260 g/l in 20 hours, when the immobilized cell concentration in the calcium alginate beads was 53.3 g dry wt./l bead volume. Ethanol yield remained almost unaffected by initial sugar concentration up to 250 g/l and was found to be about 88% of the theoretical. Maximum rate of ethanol production decreased from 22.5 g ethanol/l/h to 10.5 g ethanol/l/h while the maximum rate of total sugars utilization decreased from 74.9 g sugars/l/h to 28.5 g sugars/l/h as the initial substrate concentration was increased from 100 to 300 g/l. The concentration of free cells in the fermentation broth was low.

  12. Osteoinductive Effects of Free and Immobilized Bone Forming Peptide-1 on Human Adipose-Derived Stem Cells.

    Directory of Open Access Journals (Sweden)

    Wenyue Li

    Full Text Available Most synthetic polymeric materials currently used for bone tissue engineering lack specific signals through which cells can identify and interact with the surface, resulting in incompatibility and compromised osteogenic activity. Soluble inductive factors also have issues including a short half-live in vivo. Bone forming peptide-1 is a truncated peptide from the immature form of bone morphogenetic protein-7 (BMP-7 that displays higher osteogenic activity than full-length, mature BMP-7. In this study, we used a mussel-inspired immobilization strategy mediated by polymerization of dopamine to introduce recently discovered stimulators of bone forming peptide-1 (BFP-1 onto the surface of poly-lactic-co-glycolic acid (PLGA substrate to form a biomaterial that overcomes these challenges. Human adipose-derived stem cells (hASCs, being abundant and easy accessible, were used to test the osteogenic activity of BFP-1 and the novel biomaterial. Under osteoinductive conditions, cells treated with both BFP-1 alone and BFP-1-coated biomaterials displayed elevated expression of the osteogenic markers alkaline phosphatase (ALP, osteocalcin (OC, and RUNX2. Furthermore, hASCs associated with poly-dopamine-assisted BFP-1-immobilized PLGA (pDA-BFP-1-PLGA scaffolds promoted in vivo bone formation in nude mice. Our novel materials may hold great promise for future bone tissue engineering applications.

  13. Production of isomaltulose obtained by Erwinia sp. cells submitted to different treatments and immobilized in calcium alginate

    Directory of Open Access Journals (Sweden)

    Haroldo Yukio Kawaguti

    2011-03-01

    Full Text Available In recent decades, there has been an increase in the studies of isomaltulose obtainment, due to its physicochemical properties and physiological health benefits. These properties, which include low cariogenicity, low glycemic index and greater stability, allow the use of this sweetener as a substitute for sucrose in foods; besides the fact that it can be converted to isomalt, a dietary non-cariogenic sugar alcohol used in pharmaceuticals as well as in the food industry. Isomaltulose (6-O-α-D-glucopyronosyl-1-6-D-fructofuranose is a disaccharide reducer obtained by the enzymatic conversion of sucrose - the α-glucosyltransferase enzyme. Different treatments were performed for the preparation of whole cells; lysed cells; and crude enzyme extract of Erwinia sp. D12 strain immobilized in calcium alginate. The packed bed column of granules, containing Erwinia sp. cells sonicated and immobilized in calcium alginate (CSI, reached a maximum conversion of 53-59% sucrose into isomaltulose and it presented activity for 480 hours. The converted syrup was purified and the isomaltulose crystallization was performed through the lowering of temperature. The isomaltulose crystals presented purity of 96.5%.

  14. Preservation of Bacillus firmus Strain 37 and Optimization of Cyclodextrin Biosynthesis by Cells Immobilized on Loofa Sponge

    Directory of Open Access Journals (Sweden)

    Cristiane Moriwaki

    2012-08-01

    Full Text Available The preservation of Bacillus firmus strain 37 cells by lyophilization was evaluated and response surface methodology (RSM was used to optimize the β-cyclodextrin (β-CD production by cells immobilized on loofa sponge. Interactions were studied with the variables temperature, pH and dextrin concentration using a central composite design (CCD. Immobilization time influence on β-CD production was also investigated. B. firmus strain 37 cells remained viable after one year of storage, showing that the lyophilization is a suitable method for preservation of the microorganism. From the three-dimensional diagrams and contour plots, the best conditions for β-CD production were determined: temperature 60 °C, pH 8, and 18% dextrin. Considering that the amount of dextrin was high, a new assay was carried out, in which dextrin concentrations of 10, 15, and 18% were tested and the temperature of 60 °C and pH 8 were maintained. The results achieved showed very small differences and therefore, for economic reasons, the use of 10% dextrin is suggested. Increasing the immobilization time of cells immobilized on synthetic sponge the β-CD production decreased and did not change for cells immobilized on loofa sponge. The results of this research are important for microorganism preservation and essential in the optimization of the biosynthesis of CD.

  15. Analysis of branched DNA replication and recombination intermediates from prokaryotic cells by two-dimensional (2D) native-native agarose gel electrophoresis.

    Science.gov (United States)

    Robinson, Nicholas P

    2013-01-01

    Branched DNA molecules are generated by the essential processes of replication and recombination. Owing to their distinctive extended shapes, these intermediates migrate differently from linear double-stranded DNA under certain electrophoretic conditions. However, these branched species exist in the cell at much low abundance than the bulk linear DNA. Consequently, branched molecules cannot be visualized by conventional electrophoresis and ethidium bromide staining. Two-dimensional native-native agarose electrophoresis has therefore been developed as a method to facilitate the separation and visualization of branched replication and recombination intermediates. A wide variety of studies have employed this technique to examine branched molecules in eukaryotic, archaeal, and bacterial cells, providing valuable insights into how DNA is duplicated and repaired in all three domains of life.

  16. Hemoglobin electrophoresis

    Science.gov (United States)

    ... 12. Read More A1C test Anemia Hemoglobin Hemolytic anemia Sickle cell anemia Thalassemia Review Date 1/31/2016 Updated by: ... Anemia Blood Count Tests Blood Disorders Newborn Screening Sickle Cell Anemia Browse the Encyclopedia A.D.A.M., Inc. ...

  17. Preservation of cell viability and protein conformation on immobilization within nanofibers via electrospinning functionalized yeast.

    Science.gov (United States)

    Canbolat, M Fatih; Gera, Nimish; Tang, Christina; Monian, Brinda; Rao, Balaji M; Pourdeyhimi, Behnam; Khan, Saad A

    2013-10-09

    We investigate the immobilization of a model system of functionalized yeast that surface-display enhanced green fluorescent protein (eGFP) within chemically crosslinked polyvinyl alcohol (PVA) nanofibers. Yeast is incorporated into water insoluble nanofibrous materials by direct electrospinning with PVA followed by vapor phase chemical crosslinking of the polymer. Incorporation of yeast into the fibers is confirmed by elemental analysis and the viability is indicated by live/dead staining. Following electrospinning and crosslinking, we confirm that the yeast maintains its viability as well as the ability to express eGFP in the correct conformation. This method of processing functionalized yeast may thus be a powerful tool in the direct immobilization of properly folded, active enzymes within electrospun nanofibers with potential applications in biocatalysis.

  18. Perspectives of treatment of anemias with cells of fetal liver, immobilized in macroporous alginate-gelatin carriers

    Directory of Open Access Journals (Sweden)

    Gritsay D.V.

    2014-06-01

    Full Text Available Aim of the work was to study possibility of erythropoiesis stimulation by transplantation of fetal liver cells, seeded into macro¬porous carriers to the rats with post-hemorrhargic anemia, induced by 70% hepatectomy. Fetal liver cells (FLC were isolated from fetuses of rats with 15 days’ gestation and were cryopreserved. Decryopreserved FLC were seeded into macroporous spongy alginate-gelatin scaffolds, which were covered by alginate capsule and implanted into omentum of rats with modeled liver insufficiency. It was shown that fetal liver cells, immobilized in macroporous scaffolds after implantation have positive effect on red blood count and hemoglobin content, indicating that this approach is promising for the development of new methods of anemia treatment.

  19. Effect of immobilized collagen type IV on biological properties of endothelial cells for the enhanced endothelialization of synthetic vascular graft materials.

    Science.gov (United States)

    Heo, Yunhoe; Shin, Young Min; Lee, Yu Bin; Lim, Youn Mook; Shin, Heungsoo

    2015-10-01

    Regeneration of healthy endothelium onto vascular graft materials is imperative for prevention of intimal hyperplasia and thrombogenesis. In this study, we investigated the effect of collagen type IV (COL-IV) immobilized onto electrospun nanofibers on modulation of endothelial cell (EC) function, as a potential signal to rapid endothelialization of vascular grafts. COL-IV is assembled in basement membrane underneath intimal layer and regulates morphogenesis of blood vessels. For immobilization of COL-IV, poly(l-lactic acid) (PLLA) nanofibers (PL) were prepared as a model vascular graft substrate, onto which acrylic acid (AAc) was then grafted by using gamma-ray irradiation. AAc graft was dependent on irradiation doses and AAc concentrations, which allowed us to select the condition of 5% (v/v) AAc and 10 kGy for further conjugation of COL-IV. COL-IV immobilization was proportionally controlled as a function of its concentration. Atomic force microscope (AFM) analysis qualitatively supported immobilization of COL-IV, demonstrating increase in root mean square roughness of the PL from 665.37 ± 13.20 nm to 1440.74 ± 33.24. However, the Young's modulus of nanofibers was retained as approximately 1 MPa, regardless of surface modification. The number of ECs attached on the nanofibers with immobilized COL-IV was significantly increased by 5 times (1052 ± 138 cells/mm(2)) from pristine PL (234 ± 41 cells/mm(2)). In addition, the effect of immobilized COL-IV was profound for enhancing proliferation and up-regulation of markers implicated in rapid endothelialization. Collectively, our results suggest that COL-IV immobilized onto electrospun PLLA nanofibers may serve as a promising instructive cue used in vascular graft materials.

  20. Method for analysing glycoprotein isoforms by capillary electrophoresis

    OpenAIRE

    Frutos, Mercedes de; Díez-Masa, José Carlos; Morales-Cid, Gabriel

    2011-01-01

    [EN] The present invention relates to a new method for the purification, concentration, separation and determination of the isoforms of alpha-1-acid glycoprotein (AGP) in human blood serum samples using capillary electrophoresis. The new method is based on the immunocapture and preconcentration of the sample within the separation capillary by using an immunoadsorbent phase magnetically immobilized within the electrophoresis capillary and the subsequent desorption and separation of the glycopr...

  1. Manipulation of culture strategies to enhance capsaicin biosynthesis in suspension and immobilized cell cultures of Capsicum chinense Jacq. cv. Naga King Chili.

    Science.gov (United States)

    Kehie, Mechuselie; Kumaria, Suman; Tandon, Pramod

    2014-06-01

    Manipulation of culture strategies was adopted to study the influence of nutrient stress, pH stress and precursor feeding on the biosynthesis of capsaicin in suspension and immobilized cell cultures of C. chinense. Cells cultured in the absence of one of the four nutrients (ammonium and potassium nitrate for nitrate and potassium stress, potassium dihydrogen orthophosphate for phosphorus stress, and sucrose for sugar stress) influenced the accumulation of capsaicin. Among the stress factors studied, nitrate stress showed maximal capsaicin production on day 20 (505.9 ± 2.8 μg g(-1) f.wt) in immobilized cell, whereas in suspension cultures the maximum accumulation (345.5 ± 2.9 μg g(-1) f.wt) was obtained on day 10. Different pH affected capsaicin accumulation; enhanced accumulation of capsaicin (261.6 ± 3.4 μg g(-1) f.wt) was observed in suspension cultures at pH 6 on day 15, whereas in case of immobilized cultures the highest capsaicin content (433.3 ± 3.3 μg g(-1) f.wt) was obtained at pH 5 on day 10. Addition of capsaicin precursors and intermediates significantly enhanced the biosynthesis of capsaicin, incorporation of vanillin at 100 μM in both suspension and immobilized cell cultures resulted in maximum capsaicin content with 499.1 ± 5.5 μg g(-1) f.wt on day 20 and 1,315.3 ± 10 μg g(-1) f.wt on day 10, respectively. Among the different culture strategies adopted to enhance capsaicin biosynthesis in cell cultures of C. chinense, cells fed with vanillin resulted in the maximum capsaicin accumulation. The rate of capsaicin production was significantly higher in immobilized cells as compared to freely suspended cells.

  2. Electrophoresis device

    Science.gov (United States)

    Rhodes, P. H.; Snyder, R. S. (Inventor)

    1982-01-01

    A device for separating cellular particles of a sample substance into fractionated streams of different cellular species includes a casing having a distribution chamber, a separation chamber, and a collection chamber. The electrode chambers are separated from the separation chamber interior by means of passages such that flow variations and membrane variations around the slotted portion of the electrode chamber do not enduce flow perturbations into the laminar buffer curtain flowing in the separation chamber. The cellular particles of the sample are separated under the influence of the electrical field and the separation chamber into streams of different cellular species. The streams of separated cells enter a partition array in the collection chamber where they are fractionated and collected.

  3. Production of carboxylic acids from hydrolyzed corn meal by immobilized cell fermentation in a fibrous-bed bioreactor.

    Science.gov (United States)

    Huang, Yu Liang; Wu, Zetang; Zhang, Likun; Cheung, Chun Ming; Yang, Shang-Tian

    2002-03-01

    Corn meal hydrolyzed with amylases was used as the carbon source for producing acetic, propionic, and butyric acids via anaerobic fermentations. In this study, corn meal, containing 75% (w/w) starch, 20% (w/w) fibers, and 1.5% (w/w) protein, was first hydrolyzed using amylases at 60 degrees C. The hydrolysis yielded approximately 100% recovery of starch converted to glucose and 17.9% recovery of protein. The resulting corn meal hydrolyzate was then used, after sterilization, for fermentation studies. A co-culture of Lactococcus lactis and Clostridium formicoaceticum was used to produce acetic acid from glucose. Propionibacterium acidipropionici was used for propionic acid fermentation, and Clostridium tyrobutylicum was used for butyric acid production. These cells were immobilized on a spirally wound fibrous matrix packed in a fibrous-bed bioreactor (FBB) developed for multi-phase biological reactions or fermentation. The bioreactor was connected to a stirred-tank fermentor that provided pH and temperature controls via medium circulation. The fermentation system was operated at the recycle batch mode. Temperature and pH were controlled at 37 degrees C and 7.6, respectively, for acetic acid fermentation, 32 degrees C and 6.0, respectively, for propionic acid fermentation, and 37 degrees C and 6.0, respectively, for butyric acid production. The fermentation demonstrated a yield of approximately 100% and a volumetric productivity of approximately 1 g/(1 h) for acetic acid production. The propionic acid fermentation achieved an approximately 60% yield and a productivity of 2.12 g/(1 h), whereas the butyric acid fermentation obtained an approximately 50% yield and a productivity of 6.78 g/(1 h). These results were comparable to, or better than those fermentations using chemically defined media containing glucose as the substrate, suggesting that these carboxylic acids can be efficiently produced from direct fermentation of corn meal hydrolyzate. The corn fiber present

  4. Kinetic evaluation of nitrification performance in an immobilized cell membrane bioreactor.

    Science.gov (United States)

    Güven, D; Ubay Çokgör, E; Sözen, S; Orhon, D

    2016-01-01

    High rate membrane bioreactor (MBR) systems operated at extremely low sludge ages (superfast membrane bioreactors (SFMBRs)) are inefficient to achieve nitrogen removal, due to insufficient retention time for nitrifiers. Moreover, frequent chemical cleaning is required due to high biomass flux. This study aims to satisfy the nitrification in SFMBRs by using sponge as carriers, leading to the extension of the residence time of microorganisms. In order to test the limits of nitrification, bioreactor was run under 52, 5 and 2 days of carrier residence time (CRT), with a hydraulic retention time of 6 h. Different degrees of nitrification were obtained for different CRTs. Sponge immobilized SFMBR operation with short CRT resulted in partial nitrification indicating selective dominancy of ammonia oxidizers. At higher CRT, simultaneous nitrification-denitrification was achieved when accompanying with oxygen limitation. Process kinetics was determined through evaluation of the results by a modeling study. Nitrifier partition in the reactor was also identified by model calibration.

  5. Drying of micro-encapsulated lactic acid bacteria — Effects of trehalose and immobilization on cell survival and release properties

    Science.gov (United States)

    Li, Xiaoyan; Chen, Xiguang

    2009-03-01

    Lactic acid bacteria (LAB) were encapsulated with alginate, gelatin and trehalose additives by the extrusion method and dried at 4 °C. The microcapsules were generally spherical and had a wrinkled surface with a size of 1.7 mm ± 0.2 mm. Trehalose as a carbohydrate source in the culture medium could reduce acid production and performed no function in the positive proliferation of LAB. Using trehalose as a carbohydrate source and protective medium simultaneously had a benefit in the protection of LAB cells during the storage at 4 °C. The density of live LAB cells could be 107 CFU g-1 after 8 weeks of storage. Cells of LAB could be continuously released from the capsules from the acidic (pH 1.2) to neutral conditions (pH 6.8). The release amounts and proliferation speeds of LAB cells in neutral medium were much larger and faster than those in acidic conditions. Additionally, immobilization of LAB could improve the survival of cells when they were exposed to acidic medium (pH 1.2) with a survival rate of 76 %.

  6. Drying of Micro-Encapsulated Lactic Acid Bacteria-Effects of Trehalose and Immobilization on Cell Survival and Release Properties

    Institute of Scientific and Technical Information of China (English)

    LI Xiaoyan; CHEN Xiguang

    2009-01-01

    Lactic acid bacteria (LAB) were encapsulated with alginate, gelatin and trehalose additives by the extrusion method and dried at 4℃. The microcapsules were generally spherical and had a wrinkled surface with a size of 1.7mm±0.2mm. Trehalose as a carbohydrate source in the culture medium could reduce acid production and performed no function in the positive proliferation of LAB. Using trehalose as a carbohydrate source and protective medium simultaneously had a benefit in the protection of LAB cells during the storage at 4℃. The density of hve LAB cells could be 10- CFU g-1 after 8 weeks of storage. Cells of LAB could be con-tinuously released from the capsules from the acidic (pH 1.2) to neutral conditions (plt 6.8). The release amounts and proliferation speeds of LAB cells in neutral medium were much larger and faster than those m acidic conditions. Additionally, immobilization of LAB could improve the survival of cells when they, were exposed to acidic medium (pH 1.2) with a survival rate of 76 %.

  7. Immobilization of a Metal-Nitrogen-Carbon Catalyst on Activated Carbon with Enhanced Cathode Performance in Microbial Fuel Cells.

    Science.gov (United States)

    Yang, Wulin; Logan, Bruce E

    2016-08-23

    Applications of microbial fuel cells (MFCs) are limited in part by low power densities mainly due to cathode performance. Successful immobilization of an Fe-N-C co-catalyst on activated carbon (Fe-N-C/AC) improved the oxygen reduction reaction to nearly a four-electron transfer, compared to a twoelectron transfer achieved using AC. With acetate as the fuel, the maximum power density was 4.7±0.2 W m(-2) , which is higher than any previous report for an air-cathode MFC. With domestic wastewater as a fuel, MFCs with the Fe-N-C/AC cathode produced up to 0.8±0.03 W m(-2) , which was twice that obtained with a Pt-catalyzed cathode. The use of this Fe-N-C/AC catalyst can therefore substantially increase power production, and enable broader applications of MFCs for renewable electricity generation using waste materials.

  8. Supported Molecular Matrix Electrophoresis.

    Science.gov (United States)

    Matsuno, Yu-Ki; Kameyama, Akihiko

    2015-01-01

    Mucins are difficult to separate using conventional gel electrophoresis methods such as SDS-PAGE and agarose gel electrophoresis, owing to their large size and heterogeneity. On the other hand, cellulose acetate membrane electrophoresis can separate these molecules, but is not compatible with glycan analysis. Here, we describe a novel membrane electrophoresis technique, termed "supported molecular matrix electrophoresis" (SMME), in which a porous polyvinylidene difluoride (PVDF) membrane filter is used to achieve separation. This description includes the separation, visualization, and glycan analysis of mucins with the SMME technique.

  9. Actin Immobilization on Chitin for Purifying Myosin II: A Laboratory Exercise That Integrates Concepts of Molecular Cell Biology and Protein Chemistry

    Science.gov (United States)

    de Souza, Marcelle Gomes; Grossi, Andre Luiz; Pereira, Elisangela Lima Bastos; da Cruz, Carolina Oliveira; Mendes, Fernanda Machado; Cameron, Luiz Claudio; Paiva, Carmen Lucia Antao

    2008-01-01

    This article presents our experience on teaching biochemical sciences through an innovative approach that integrates concepts of molecular cell biology and protein chemistry. This original laboratory exercise is based on the preparation of an affinity chromatography column containing F-actin molecules immobilized on chitin particles for purifying…

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

    Science.gov (United States)

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

    2016-08-10

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

  11. Effects of initial pH value of the medium on the alcoholic fermentation performance of Saccharomyces cerevisiae cells immobilized on nipa leaf sheath pieces

    Directory of Open Access Journals (Sweden)

    Hoang Duc Toan Le

    2014-12-01

    Full Text Available Immobilized yeast on nipa leaf sheath pieces was applied to ethanol fermentation using the medium with different initial pH values (5.1, 4.5, 4.0, and 3.5. Control samples with the free yeast were also carried out under the same conditions. Low pH value of 4.0 or 3.5 significantly reduced yeast growth and increased the residual sugar level in the fermentation broths for both the immobilized and free cells. In all cases, the ethanol content produced and ethanol formation rate of the immobilized yeast were 13-33% and 35-69%, respectively, higher than those of the free yeast. In addition, the residual sugar content in the immobilized yeast cultures was 2.1-20.5 times lower than that in the free yeast cultures. The yeast immobilized on nipa leaf stem pieces exhibited higher alcoholic fermentation performance than the free yeast in medium with low pH value. This support was potential for further research for application in ethanol industry.

  12. The microalga Chlamydomonas reinhardtii CW-15 as a solar cell for hydrogen peroxide photoproduction. Comparison between free and immobilized cells and thylakoids for energy conversion efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Scholz, W.; Galvan, F.; Rosa, F.F. de la [Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla y CSIC, Sevilla (Spain)

    1995-11-28

    Immobilized cells and thylakoid vesicles of the microalga Chlamydomonas reinhardtii CW-15 have been developed as a solar cell because of their capabilities of producing hydrogen peroxide. This compound is an efficient and clean fuel used for rocket propulsion, motors and for heating. Hydrogen peroxide is produced by the photosystem in a catalyst cycle in which a redox mediator (methyl viologen) is reduced by electrons obtained from water by the photosynthetic apparatus of the microalga and it is re-oxidized by the oxygen dissolved in the solution. The photoproduction has been investigated using a discontinuous system with whole cells, or thylakoid vesicles, free or immobilized on alginate. The stimulation by azide as an inhibitor of catalase has also been analyzed. Under determined optimum conditions, the photoproduction by Ca-alginate entrapped cells, with a rate of 33 {mu}mol H{sub 2}O{sub 2}/mg Chl.h, was maintained for several hours with an energy conversion efficiency of 0.25%

  13. Diagnosis of cutaneous T-cell lymphoma detecting T-cell receptor gamma chain gene monoclonality by denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Lapière, K; Dhaene, K; Matthieu, L; Hübner, R; Lambert, J; Van Marck, E

    1999-04-01

    Cutaneous T-cell lymphomas represent a group of malignant lymphoproliferative disorders characterised by the occurrence of a monoclonal population of T-lymphocytes. Diagnosis of early stages of this disease is a difficult challenge for both the dermatologist and the dermatopathologist. With the aid of the polymerase chain reaction it is possible to amplify specific regions of the T-cell receptor gamma gene. The amplification products can then be separated by denaturing gradient gel electrophoresis in order to detect a monoclonal population of T-lymphocytes in the infiltrate. We studied 4 patients with the clinicopathologic diagnosis of mycosis fungoides and 2 patients diagnosed as large plaque parapsoriasis. A monoclonal population was detected in 3 of the 4 mycosis fungoides cases and in 1 of the patients with large plaque parapsoriasis. This indicates that our analysis can help us establishing a diagnosis, and it can also help us to identify patients with a possible early stage of the disease, which clinically or histologically is not yet recognised as such.

  14. Applications of capillary electrophoresis and laser-induced fluorescence detection to the analysis of trace species: From single cells to single molecules

    Energy Technology Data Exchange (ETDEWEB)

    Qifeng, X.

    1995-11-01

    This Ph.D. Thesis describes several separation and detection schemes for the analysis of small volume and amount of samples, such as intracellular components and single enzymes developed during research. Indirect Laser-induced fluorescence detection and capillary electrophoresis were used to quantify lactate and pyruvate in single red blood cells. The assay of specific enzyme activities was achieved by monitoring the highly fluorescent enzymatic reaction product, NADH. LDH activity was found not to be a unique marker for diagnosis of leukemia. Reactions of single LDH-1 molecules were investigated by monitoring the reaction product with LIF detection.

  15. Direct methods for dynamic monitoring of secretions from single cells by capillary electrophoresis and microscopy with laser-induced native fluorescence detection

    Energy Technology Data Exchange (ETDEWEB)

    Tong, Wei [Iowa State Univ., Ames, IA (United States)

    1997-10-08

    Microscale separation and detection methods for real-time monitoring of dynamic cellular processes (e.g., secretion) by capillary electrophoresis (CE) and microscopic imaging were developed. Ultraviolet laser-induced native fluorescence (LINF) provides simple, sensitive and direct detection of neurotransmitters and proteins without any derivatization. An on-column CE-LINF protocol for quantification of the release from single cell was demonstrated. Quantitative measurements of both the amount of insulin released from and the amount remaining in the cell (βTC3) were achieved simultaneously. Secretion of catecholamines (norepinephrine (NE) and epinephrine (E)) from individual bovine adrenal chromaffin cells was determined using the on-column CE-LINF. Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved by LINF imaging microscopy with high temporal and spatial resolution. The secretion of serotonin from individual leech Retzius neurons was directly characterized by LINF microscopy with high spatial resolution.

  16. Direct methods for dynamic monitoring of secretions from single cells by capillary electrophoresis and microscopy with laser-induced native fluorescence detection

    Energy Technology Data Exchange (ETDEWEB)

    Tong, W.

    1997-10-08

    Microscale separation and detection methods for real-time monitoring of dynamic cellular processes (e.g., secretion) by capillary electrophoresis (CE) and microscopic imaging were developed. Ultraviolet laser-induced native fluorescence (LINF) provides simple, sensitive and direct detection of neurotransmitters and proteins without any derivatization. An on-column CE-LINF protocol for quantification of the release from single cell was demonstrated. Quantitative measurements of both the amount of insulin released from and the amount remaining in the cell ({beta}TC3) were achieved simultaneously. Secretion of catecholamines (norepinephrine (NE) and epinephrine (E)) from individual bovine adrenal chromaffin cells was determined using the on-column CE-LINF. Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved by LINF imaging microscopy with high temporal and spatial resolution. The secretion of serotonin from individual leech Retzius neurons was directly characterized by LINF microscopy with high spatial resolution.

  17. Two-dimensional gel electrophoresis analysis of mycelial cells treated with Tween 80: differentially expressed protein related to enhanced metabolite production.

    Science.gov (United States)

    Zhang, Bo-Bo; Chen, Lei; Cheung, Peter C K

    2012-10-24

    Two-dimensional gel electrophoresis identified 40 differentially expressed proteins which explained the mechanisms underlying the stimulatory effect of Tween 80 for exopolysaccharide production in the mycelium of an edible mushroom Pleurotus tuber-regium. The up-regulation of fatty acid synthase alpha subunit FasA might promote the synthesis of long-chain fatty acids and their incorporation into the mycelial cell membranes, increasing the membrane permeability. A down-regulation of Phospholipase D1 and an up-regulation of Hypothetical protein PGUG_02954 might mediate signal transduction between the mycelial cells and the extracellular stimulus (Tween 80). The down-regulated ATP-binding cassette transporter protein might function as pumps to extrude exopolysaccharide out of the cells that lead to a significant increase in its production. The present results explained how stimulatory agents like Tween 80 can increase mycelial cell membrane permeability to enhance the production of useful extracellular metabolites by submerged fermentation.

  18. Immobilization of cross linked Col-I–OPN bone matrix protein on aminolysed PCL surfaces enhances initial biocompatibility of human adipogenic mesenchymal stem cells (hADMSC)

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young-Hee; Jyoti, Md. Anirban; Song, Ho-Yeon, E-mail: songmic@sch.ac.kr

    2014-06-01

    In bone tissue engineering surface modification is considered as one of the important ways of fabricating successful biocompatible material. Addition of biologically active functionality on the surfaces has been tried for improving the overall biocompatibility of the system. In this study poly-ε-caprolactone film surfaces have been modified through aminolysis and immobilization process. Collagen type I (COL-I) and osteopontin (OPN), which play an important role in osteogenesis, was immobilized onto PCL films followed by aminolysis treatment using 1,6-hexanediamine. Characterization of animolysed and immobilized surfaces were done by a number techniques using scanning electron microscopy (SEM), FT-IR, XPS, ninhydrin staining, SDS-PAGE and confocal microscopy and compared between the modified and un-modified surfaces. Results of the successive experiments showed that aminolysis treatment was homogeneously achieved which helped to entrap or immobilize Col-I–OPN proteins on surfaces of PCL film. In vitro studies with human adipogenic mesenchymal stem cells (hADMSC) also confirmed the attachment and proliferation of cells was better in modified PCL surfaces than the unmodified surfaces. SEM, confocal microscopy and MTT assay showed a significant increase in cell spreading, attachment and proliferations on the biofunctionalized surfaces compared to the unmodified PCL surfaces at all-time points indicating the success of surface biofunctionalization.

  19. Fast methods for analysis of neurotransmitters from single cell and monitoring their releases in central nervous system by capillary electrophoresis, fluorescence microscopy and luminescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ziqiang

    1999-12-10

    Fast methods for separation and detection of important neurotransmitters and the releases in central nervous system (CNS) were developed. Enzyme based immunoassay combined with capillary electrophoresis was used to analyze the contents of amino acid neurotransmitters from single neuron cells. The release of amino acid neurotransmitters from neuron cultures was monitored by laser induced fluorescence imaging method. The release and signal transduction of adenosine triphosphate (ATP) in CNS was studied with sensitive luminescence imaging method. A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Detection was based on monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. The detection limit of glutamate is down to 10{sup {minus}8} M level, which is 1 order of magnitude lower than the previously reported detection limit based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most of amino acids. The glutamate content in single human erythrocyte and baby rat brain neurons were determined with this method and results agreed well with literature values.

  20. Fast methods for analysis of neurotransmitters from single cell and monitoring their releases in central nervous system by capillary electrophoresis, fluorescence microscopy and luminescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ziqiang [Iowa State Univ., Ames, IA (United States)

    1999-12-10

    Fast methods for separation and detection of important neurotransmitters and the releases in central nervous system (CNS) were developed. Enzyme based immunoassay combined with capillary electrophoresis was used to analyze the contents of amino acid neurotransmitters from single neuron cells. The release of amino acid neurotransmitters from neuron cultures was monitored by laser induced fluorescence imaging method. The release and signal transduction of adenosine triphosphate (ATP) in CNS was studied with sensitive luminescence imaging method. A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Detection was based on monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. The detection limit of glutamate is down to 10-8 M level, which is 1 order of magnitude lower than the previously reported detection limit based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most of amino acids. The glutamate content in single human erythrocyte and baby rat brain neurons were determined with this method and results agreed well with literature values.

  1. Nitrogenase activity of immobilized Azotobacter

    Energy Technology Data Exchange (ETDEWEB)

    Seyhan, E.; Kirwan, D.J.

    1979-02-01

    As part of a program to investigate the use of biological nitrogen fixation for fertilizer ammonia production, an investigation into the immobilization of the aerobic, nitrogen-fixing bacterium, Azotobacter vinelandii was undertaken. Immobilization was accomplished by adsorption onto an anionic exchange cellulose (Cellex E) with loadings as high as 10/sup 11/ cells/g resin. Immobilized cell preparations were tested under both batch and continuous-flow conditions. Nitrogenease activities as high as 4200 nmol/min g resin were observed as measured by the acetylene reduction assay. Immobilized cells retained their activity for as long as 117 hr in a continuous-flow reactor. Activity loss appeared to be related to the development of a variant strain.

  2. Optimization of date syrup for enhancement of the production of citric acid using immobilized cells of Aspergillus niger.

    Science.gov (United States)

    Mostafa, Yasser S; Alamri, Saad A

    2012-04-01

    Date syrup as an economical source of carbohydrates and immobilized Aspergillus niger J4, which was entrapped in calcium alginate pellets, were employed for enhancing the production of citric acid. Maximum production was achieved by pre-treating date syrup with 1.5% tricalcium phosphate to remove heavy metals. The production of citric acid using a pretreated medium was 38.87% higher than an untreated one that consumed sugar. The appropriate presence of nitrogen, phosphate and magnesium appeared to be important in order for citric acid to accumulate. The production of citric acid and the consumed sugar was higher when using 0.1% ammonium nitrate as the best source of nitrogen. The production of citric acid increased significantly when 0.1 g/l of KH2PO4 was added to the medium of date syrup. The addition of magnesium sulfate at the rate of 0.20 g/l had a stimulating effect on the production of citric acid. Maximum production of citric acid was obtained when calcium chloride was absent. One of the most important benefits of immobilized cells is their ability and stability to produce citric acid under a repeated batch culture. Over four repeated batches, the production of citric acid production was maintained for 24 days when each cycle continued for 144 h. The results obtained in the repeated batch cultivation using date syrup confirmed that date syrup could be used as a medium for the industrial production of citric acid.

  3. Alkali-Soluble Pectin Is the Primary Target of Aluminum Immobilization in Root Border Cells of Pea (Pisum sativum).

    Science.gov (United States)

    Yang, Jin; Qu, Mei; Fang, Jing; Shen, Ren Fang; Feng, Ying Ming; Liu, Jia You; Bian, Jian Feng; Wu, Li Shu; He, Yong Ming; Yu, Min

    2016-01-01

    We investigated the hypothesis that a discrepancy of Al binding in cell wall constituents determines Al mobility in root border cells (RBCs) of pea (Pisum sativum), which provides protection for RBCs and root apices under Al toxicity. Plants of pea (P. sativum L. 'Zhongwan no. 6') were subjected to Al treatments under mist culture. The concentration of Al in RBCs was much higher than that in the root apex. The Al content in RBCs surrounding one root apex (10(4) RBCs) was approximately 24.5% of the total Al in the root apex (0-2.5 mm), indicating a shielding role of RBCs for the root apex under Al toxicity. Cell wall analysis showed that Al accumulated predominantly in alkali-soluble pectin (pectin 2) of RBCs. This could be attributed to a significant increase of uronic acids under Al toxicity, higher capacity of Al adsorption in pectin 2 [5.3-fold higher than that of chelate-soluble pectin (pectin 1)], and lower ratio of Al desorption from pectin 2 (8.5%) compared with pectin 1 (68.5%). These results indicate that pectin 2 is the primary target of Al immobilization in RBCs of pea, which impairs Al access to the intracellular space of RBCs and mobility to root apices, and therefore protects root apices and RBCs from Al toxicity.

  4. Alkali-Soluble Pectin Is the Primary Target of Aluminum Immobilization in Root Border Cells of Pea (Pisum sativum)

    Science.gov (United States)

    Yang, Jin; Qu, Mei; Fang, Jing; Shen, Ren Fang; Feng, Ying Ming; Liu, Jia You; Bian, Jian Feng; Wu, Li Shu; He, Yong Ming; Yu, Min

    2016-01-01

    We investigated the hypothesis that a discrepancy of Al binding in cell wall constituents determines Al mobility in root border cells (RBCs) of pea (Pisum sativum), which provides protection for RBCs and root apices under Al toxicity. Plants of pea (P. sativum L. ‘Zhongwan no. 6’) were subjected to Al treatments under mist culture. The concentration of Al in RBCs was much higher than that in the root apex. The Al content in RBCs surrounding one root apex (104 RBCs) was approximately 24.5% of the total Al in the root apex (0–2.5 mm), indicating a shielding role of RBCs for the root apex under Al toxicity. Cell wall analysis showed that Al accumulated predominantly in alkali-soluble pectin (pectin 2) of RBCs. This could be attributed to a significant increase of uronic acids under Al toxicity, higher capacity of Al adsorption in pectin 2 [5.3-fold higher than that of chelate-soluble pectin (pectin 1)], and lower ratio of Al desorption from pectin 2 (8.5%) compared with pectin 1 (68.5%). These results indicate that pectin 2 is the primary target of Al immobilization in RBCs of pea, which impairs Al access to the intracellular space of RBCs and mobility to root apices, and therefore protects root apices and RBCs from Al toxicity. PMID:27679639

  5. Proteins pattern alteration in AZT-treated K562 cells detected by two-dimensional gel electrophoresis and peptide mass fingerprinting

    Directory of Open Access Journals (Sweden)

    Mignogna Giuseppina

    2006-03-01

    Full Text Available Abstract In this study we report the effect of AZT on the whole protein expression profile both in the control and the AZT-treated K562 cells, evidenced by two-dimensional gel electrophoresis and peptide mass fingerprinting analysis. Two-dimensional gels computer digital image analysis showed two spots that appeared up-regulated in AZT-treated cells and one spot present only in the drug exposed samples. Upon extraction and analysis by peptide mass fingerprinting, the first two spots were identified as PDI-A3 and stathmin, while the third one was proved to be NDPK-A. Conversely, two protein spots were present only in the untreated K562 cells, and were identified as SOD1 and HSP-60, respectively.

  6. Immobilization of Trichosporon cutaneum R 57 Cells onto Methylcellulose/SiO2 Hybrids and Biosorption of Cadmium and Copper Ions

    Directory of Open Access Journals (Sweden)

    Georgieva N.

    2009-12-01

    Full Text Available Methylcellulose/Silica (MC/SiO2 hybrids were synthesized via poly step sol-gel method. SiO2 was included into the hybrids from two silica precursors - methyltriethoxysilane (MTES and ethyltrimethoxysilane (ETMS with different quantity of organic part-5, 20 and 50 wt.%. The filamentous yeasts Trichosporon cutaneum strain R 57 was immobilized onto the synthesized MC/SiO2 hybrids. After immobilization the hybrid materials were used in the processes of sorption of cadmium and copper ions. The obtained results of protein content analysis indicated that the amount of protein increased with increasing of MC in the hybrids. It was established that the maximal efficiency of copper and cadmium removal were observed for hybrid materials containing MTES and 50 wt.% MC - 66% and 26% respectively. For ETMS and 50 wt.% MC a high value of copper removal was 56% and for cadmium - 45% removal, respectively. FTIR analysis of free and immobilized cells with metal ions was conducted. SEM images showed successful immobilization of the yeasts cells. Second order model was employed in order to investigate the kinetics of copper and cadmium biosorption.

  7. Evaluation of DNA damage in oral precancerous and squamous cell carcinoma patients by single cell gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Sanjit Mukherjee

    2011-01-01

    Materials and Methods: Peripheral blood was collected by venepuncture and comet assay was performed using SCGE. Mean tail length was compared between diagnostic groups and between different oral habit groups using t-tests and analysis of variance (ANOVA. Pearson′s product moment correlation was used to examine the linear association between the extent of DNA damage and oral habit pack-years. Scheffe′s pair-wise test was employed to adjust for multiple comparisons. Results: None of the controls were associated with any oral habits. Mean (±SD tail lengths (in mm for cancer (24.95 ± 5.09 and leukoplakia (12.96 ± 2.68 were significantly greater than in controls (8.54 ± 2.55, P<0.05. After adjustment, well-, moderately, and poorly differentiated carcinomas had significantly greater tail length than controls. Whereas the extent of DNA damage in cancer cases was significantly greater in leukoplakia than in compared to OSMF (11.03 ± 5.92, the DNA damage in latter was not different from controls. DNA damage for people with any oral habit (19.78 ± 7.77 was significantly greater than those with no habits (8.54 ± 2.55; P<0.0001. Conclusions: DNA damage measured by SCGE is greater in leukoplakia and squamous cell carcinoma, but not in OSMF. Deleterious oral habits are also associated with greater DNA damage.

  8. Comparative study of bio-ethanol production from mahula (Madhuca latifolia L.) flowers by Saccharomyces cerevisiae cells immobilized in agar agar and Ca-alginate matrices

    Energy Technology Data Exchange (ETDEWEB)

    Behera, Shuvashish; Mohanty, Rama Chandra [Department of Botany, Utkal University, Vani Vihar, Bhubaneswar 751004, Orissa (India); Kar, Shaktimay; Ray, Ramesh Chandra [Microbiology Laboratory, Central Tuber Crops Research Institute (Regional Centre), Bhubaneswar 751019, Orissa (India)

    2010-01-15

    Batch fermentation of mahula (Madhuca latifolia L., a tree commonly found in tropical rain forest) flowers was carried out using immobilized cells (in agar agar and calcium alginate) and free cells of Saccharomyces cerevisiae. The ethanol yields were 151.2, 154.5 and 149.1 g kg{sup -1} flowers using immobilized (in agar agar and calcium alginate) and free cells, respectively. Cell entrapment in calcium alginate was found to be marginally superior to those in agar agar (2.2% more) as well as over free cell (3.5% more) as regard to ethanol yield from mahula flowers is concerned. Further, the immobilized cells were physiologically active at least for three cycles [150.6, 148.5 and 146.5 g kg{sup -1} (agar agar) and 152.8, 151.5 and 149.5 g kg{sup -1} flowers (calcium alginate) for first, second and third cycle, respectively] of ethanol fermentation without apparently lowering the productivity. Mahula flowers, a renewable, non-food-grade cheap carbohydrate substrate from non-agricultural environment such as forest can serve as an alternative to food grade sugar/starchy crops such as maize, sugarcane for bio-ethanol production. (author)

  9. The application of single cell gel electrophoresis or comet assay to human monitoring studies Aplicacion de la electroforesis unicelular o ensayo cometa en estudios de monitoreo humano

    Directory of Open Access Journals (Sweden)

    Mahara Valverde

    1999-11-01

    Full Text Available Objective. In the search of new human genotoxic biomarkers, the single cell gel electrophoresis assay has been proposed as a sensible alternative. Material and methods. This technique detects principally single strand breaks as well as alkali-labile and repair-retarded sites. Results. Herein we present our experience using the single cell gel electrophoresis assay in human population studies, both occupationally and environmentally exposed. Conclusions. We discuss the assay feasibility as a genotoxic biomarker.Objetivo. En la búsqueda de nuevos marcadores genotóxicos aplicables a estudios de poblaciones humanas expuestos a xenobióticos, la utilización del ensayo de electroforesis en una sola célula se ha propuesto como un método sensible y una buena alternativa. Material y métodos. Esta técnica detecta rompimientos en el ADN de cadena sencilla, así como sitios álcali lábiles y sitios retardados de reparación. Resultados. En este trabajo, presentamos nuestra experiencia utilizando este ensayo en poblaciones humanas expuestas ocupacionalmente o ambientalmente a diferentes xenobióticos. Conclusiones. Se discute la posible utilidad de este ensayo como un biomarcador de efecto genotóxico.

  10. Affinity in electrophoresis.

    Science.gov (United States)

    Heegaard, Niels H H

    2009-06-01

    The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

  11. AN INTEGRATIVE WAY OF TEACHING MOLECULAR CELL BIOLOGY AND PROTEIN CHEMISTRY USING ACTIN IMMOBILIZATION ON CHITIN FOR PURIFYING MYOSIN II.

    Directory of Open Access Journals (Sweden)

    M.G. Souza

    2007-05-01

    Full Text Available Our intent is to present our experience on teaching Molecular Cell Biology andProtein Chemistry at UNIRIO through an innovative approach that includes myosin IIextraction and purification. We took advantage of the properties of muscle contractionand propose a simple method for purifying myosin II by affinity chromatography. Thisoriginal method is based on the preparation of an affinity column containing actinmolecules covalently bound to chitin particles. We propose a three-week syllabus thatincludes lectures and bench experimental work. The syllabus favors the activelearning of protein extraction and purification, as well as, of scientific concepts suchas muscle contraction, cytoskeleton structure and its importance for the living cell. Italso promotes the learning of the biotechnological applications of chitin and theapplications of protein immobilization in different industrial fields. Furthermore, theactivities also target the development of laboratorial technical abilities, thedevelopment of problem solving skills and the ability to write up a scientific reportfollowing the model of a scientific article. It is very important to mention that thissyllabus can be used even in places where a facility such as ultra-centrifugation islacking.

  12. Effect of temperature and pH on ethanol production by free and immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract

    Energy Technology Data Exchange (ETDEWEB)

    Bajpai, P.; Margaritis, A.

    1987-01-01

    The effect of temperature and pH on the kinetics of ethanol production by free and calcium alginate immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract was investigated. With the free cells, the ethanol and biomass yields were relatively constant over the temperature range 25-35 degrees C, but dropped sharply beyond 35 degrees C. Other kinetic parameters, specific growth rate, specific ethanol production rate, and specific total sugar uptake rate were maximum at 35 degrees C. However, with the immobilized cells, ethanol yield remained almost constant in the temperatue range 25-45 degrees C, and the specific ethanol production rate and specific total sugar uptake rate attained their maximum values at 40 degrees C. For the pH range between 3 and 7, the free-cell optimum for growth and product formation was found to be circa pH 5. At this pH, the specific growth rate was 0.35/h and specific ethanol production rate was 2.83 g/g/h. At values higher or lower than pH 5, a sharp decrease in specific ethanol production rate as well as specific growth rate was observed. In comparison, the immobilized cells showed a broad optimum pH profile. The best ethanol production rates were observed between pH 4 and 6. (Refs. 22).

  13. A process for the treatment of olive mill waste waters by immobilized cells.

    Directory of Open Access Journals (Sweden)

    ElYachioui, M.

    2005-06-01

    Full Text Available Mould strains were immobilized on sawdust from woods as a solid material for the treatment of Olive Mill Waste (OMW waters. Assays were carried out in flasks. The treatment process was monitored by physico-chemical determinations including pH, polyphenols and COD, which were followed up during the incubation time. In parallel the chemical inhibitory activity of OMW was confirmed biologically by the determination of some microorganisms in the medium including the plate count, yeasts and lactic acid bacteria. Results indicated that the polyphenol degradation level was 87 %. The COD was also reduced by 60 %. The pH of the effluent increased from 4.5 to 6.6. The microbial profiles showed their best growth during the treatment period indicating a removal of the inhibitory activities from the OMW waters. The growth patterns of all microorganism groups were similar and could reach high levels in the effluent.Cepas de moho fueron inmovilizadas sobre serrín de madera como material sólido para el tratamiento de aguas residuales de un molino de aceituna (OMW. Los ensayos se realizaron en matraces. El proceso de tratamiento se monitorizó mediante determinaciones físico-químicas incluyendo pH, polifenoles y DQO, que también se analizaron durante el tiempo de incubación. En paralelo, la actividad inhibidora química de las OMW se confirma biológicamente mediante su efecto sobre algunos microorganismos incluyendo levaduras y bactérias ácido lácticas. Los resultados indicaron que los polifenoles se degradan hasta un nivel del 87 %. La DQO se redujo también al 60 %. El pH del efluente aumentó de 4.5 a 6.6. Los perfiles microbiológicos mostraron un mejor crecimiento a medida que avanzaba el tratamiento indicando una supresión de las actividades inhibidoras de las aguas (OMW. El comportamiento del crecimiento de todos los grupos de microorganismos fue similar y puede alcanzar altos niveles en el efluente

  14. Electrophoresis for biological production

    Science.gov (United States)

    Mccreight, L. R.

    1977-01-01

    Preparative electrophoresis may provide a unique method for meeting ever more stringent purity requirements. Prolonged near zero gravity in space may permit the operation of preparative electrophoresis equipment with 100 times greater throughput than is currently available. Some experiments with influenza Virus Antigen, Erythropoietin and Antihemophaliac Factor, along with process and economic projections, are briefly reviewed.

  15. Asymmetric Reduction of3,5-Bistrifluoromethyl Acetophenone with NADH Regeneration by Immobilized Cells of Saccharomyces rhodotorula in Aqueous-Organic Solvent Biphasic System%Asymmetric Reduction of 3,5-Bistrifluoromethyl Acetophenone with NADH Regeneration by Immobilized Cells of Saccharomyces rhodotorula in Aqueous-Organic Solvent Biphasic System

    Institute of Scientific and Technical Information of China (English)

    ZHANGFang; XUEYing; LILi; WANG Min

    2011-01-01

    Asymmetric reduction of 3,5-bistrifluoromethyl acetophenone to produce (S)-3,5-bistrifluoromethylphenyl ethanol was successfully carried out with sodium alginate immobilized Saccharomyces rhodotorula cells in an aqueous-organic solvent biphasic system.The possible influential factors were examined thoroughly according to their effects on conversion rate and e.e of the product.Organic solvents were rated by their biocompatibility and conversion potential.The immobilized cells [125 mg/mL in 20 mmol/L Tris-HCl buffer and 5%(φ) octane at pH 8] showed the best conversion with a substrate concentration of 1.42 g/L at 30C with glucose as co-substrate for cofactor regeneration.Sequential 8-batch process was carried out with immobilized cells with a slow decrease in conversion and e.e.The immobilized cells showed stable catalytic activity with 50% reserved activity and are superior especially in reusability in comparison with resting cells.

  16. Effect of DNA damage caused by cryopreservation of spermatozoa using a modified Single cell gell electrophoresis in the freshwater catfish Pangasianodon hypophthalmus (Fowler, 1936

    Directory of Open Access Journals (Sweden)

    Kuppusamy Umaa Rani

    2014-07-01

    Full Text Available Objective: To detect the integrity of sperm DNA in the catfish Pangasianodon hypophthalmus cryopreserved with Hanks balanced salt solution, 5%-30% dimethyl acetamide (DMA using the single-cell gel electrophoresis in order to test how sperm cryopreservation affected nuclear DNA stability. Methods: Electrophoresis was conducted for 60 min at 130 mA and 15 V. The comet images were analyzed using software CometScore 1.5, and parameters such as comet length, tail length and percentage DNA in the tail were obtained. Then the comet rate and damage coefficient were calculated. Results: There were no significant differences in motility, comet rate and damage coefficient between fresh sperm and cryopreserved sperm stored in 5%, 10%, 15% and 20% DMA, while the sperm cryopreserved with 25% and 30% DMA had lower motility, higher comet length and damage coefficients than those of fresh sperm. Conclusions: There was a positive correlation between comet rate of cryopreserved sperm and the concentration of DMA. The toxicity of cryoprotectant is the main factor for DNA damage in cryopreserved sperm.

  17. Binary Oscillatory Crossflow Electrophoresis

    Science.gov (United States)

    Molloy, Richard F.; Gallagher, Christopher T.; Leighton, David T., Jr.

    1997-01-01

    Electrophoresis has long been recognized as an effective analytic technique for the separation of proteins and other charged species, however attempts at scaling up to accommodate commercial volumes have met with limited success. In this report we describe a novel electrophoretic separation technique - Binary Oscillatory Crossflow Electrophoresis (BOCE). Numerical simulations indicate that the technique has the potential for preparative scale throughputs with high resolution, while simultaneously avoiding many problems common to conventional electrophoresis. The technique utilizes the interaction of an oscillatory electric field and a transverse oscillatory shear flow to create an active binary filter for the separation of charged protein species. An oscillatory electric field is applied across the narrow gap of a rectangular channel inducing a periodic motion of charged protein species. The amplitude of this motion depends on the dimensionless electrophoretic mobility, alpha = E(sub o)mu/(omega)d, where E(sub o) is the amplitude of the electric field oscillations, mu is the dimensional mobility, omega is the angular frequency of oscillation and d is the channel gap width. An oscillatory shear flow is induced along the length of the channel resulting in the separation of species with different mobilities. We present a model that predicts the oscillatory behavior of charged species and allows estimation of both the magnitude of the induced convective velocity and the effective diffusivity as a function of a in infinitely long channels. Numerical results indicate that in addition to the mobility dependence, the steady state behavior of solute species may be strongly affected by oscillating fluid into and out of the active electric field region at the ends of the cell. The effect is most pronounced using time dependent shear flows of the same frequency (cos((omega)t)) flow mode) as the electric field oscillations. Under such conditions, experiments indicate that

  18. An automated method for determining the cytoadhesion of Plasmodium falciparum-infected erythrocytes to immobilized cells

    DEFF Research Database (Denmark)

    Hempel, Casper; Boisen, Ida M; Efunshile, Akinwale;

    2015-01-01

    BACKGROUND: Plasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature. This is central in malaria pathogenesis but in vitro studies of cytoadhesion rely mainly on manual counting methods. The current study aimed at developing...... an automated high-throughput method for this purpose utilizing the pseudoperoxidase activity of intra-erythrocytic haemoglobin. METHODS: Chinese hamster ovary (CHO) cells were grown to confluence in chamber slides and microtiter plates. Cytoadhesion of co-cultured P. falciparum, selected for binding to CHO...... using: i) binding of P. falciparum-infected erythrocytes to CHO cells over-expressing chondroitin sulfate A and ii) CHO cells transfected with CD36. Binding of infected erythrocytes including field isolates to primary endothelial cells was also performed. Data was analysed using linear regression...

  19. Production of non-alcoholic beer using free and immobilized cells of Saccharomyces cerevisiae deficient in the tricarboxylic acid cycle.

    Science.gov (United States)

    Navrátil, Marián; Dömény, Zoltán; Sturdík, Ernest; Smogrovicová, Daniela; Gemeiner, Peter

    2002-04-01

    Production of non-alcoholic beer using Saccharomyces cerevisiae has been studied. Non-recombinant mutant strains with a defect in the synthesis of tricarboxylic-acid-cycle enzymes were used and applied in both free and pectate-immobilized form, using both batch and packed-bed continuous systems. After fermentation, basic parameters of the beer produced by five mutant strains were compared with a standard strain of brewing yeast. Results showed that the beer prepared by mutant yeast cells was characterized by lower levels of total alcohols, with ethanol concentrations between 0.07 and 0.31% (w/w). The organic acids produced, especially lactic acid, in concentrations up to 1.38 g x l(-1) had a strong protective effect on the microbial stability of the final product and thus the usual addition of lactic acid could be omitted. Application of the yeast mutants appears to be a good alternative to the classical methods for the production of non-alcoholic beer.

  20. Removal of Di-w-butyl Phthalate Using Immobilized Microbial Cells%固定化微生物细胞降解邻苯二甲酸二丁酯

    Institute of Scientific and Technical Information of China (English)

    孙晓然; 单忠键

    2007-01-01

    The biodegradation of di-n-butyl phthalate (DBF) using immobilized microbial cells was carried out in an internal airlift loop reactor with ceramic honeycomb supports. A strain that is capable of degrading DBF was isolated from the activated sludge and identified as Bacillus sp. using 16S rDNA sequential analysis. Bacillus sp. could be rapidly attached onto the ceramic honeycomb supports. The immobilized cells could effectively degrade DBF in batch and continuous experiments. When the influent concentration of DBF was 50mg·L-1, the effluent DBF reached less than 1mg·L-1 with 6h hydraulic retention time (HRT) in continuous experiment. The immobilized microbial cells could grow and accumulate through the biodegradation of DBF, and the rate of degradation is accordingly increased. The possible pathway of DBF biodegradation using immobilized cells was tentatively proposed.

  1. Affinity Capillary Electrophoresis:Study of the Binding of HIV-1 gp41 with a Membrane Protein (P45) on the Human B Cell Line,Raji

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Affinity capillary electrophoresis has been used to study the interaction between a membrane protein (P45) isolated from the Human B cell line, Raji, and rsgp41. P45, rsgp41 and the complexes were well resolved. The entire separation was achieved in less than 3min. Formations of two kinds of stable P45-rsgp41 complexes were confirmed based on migration time comparison; the binding equilibrium was achieved as soon as two proteins were mixed. The results indicate that the interaction between P45 and rsgp41 is strong with a fast association rate and a slow dissociation rate, and there are at least two kinds of binding sites with different binding constants between P45 and rsgp41.

  2. Determination of NAD+ and NADH level in a Single Cell Under H2O2 Stress by Capillary Electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Xi, Wenjun [Iowa State Univ., Ames, IA (United States)

    2008-01-01

    A capillary electrophoresis (CE) method is developed to determine both NAD+ and NADH levels in a single cell, based on an enzymatic cycling reaction. The detection limit can reach down to 0.2 amol NAD+ and 1 amol NADH on a home-made CE-LIF setup. The method showed good reproducibility and specificity. After an intact cell was injected into the inlet of a capillary and lysed using a Tesla coil, intracellular NAD+ and NADH were separated, incubated with the cycling buffer, and quantified by the amount of fluorescent product generated. NADH and NAD+ levels of single cells of three cell lines and primary astrocyte culture were determined using this method. Comparing cellular NAD+ and NADH levels with and without exposure to oxidative stress induced by H2O2, it was found that H9c2 cells respond to the stress by reducing both cellular NAD+ and NADH levels, while astrocytes respond by increasing cellular NADH/NAD+ ratio.

  3. Electrophoresis release of hemoglobin from living red blood cells%活体红细胞内血红蛋白的电泳释放

    Institute of Scientific and Technical Information of China (English)

    秦文斌

    2011-01-01

    当今的蛋白质组学研究,都是先裂解细胞放出蛋白质,然后对蛋白质溶液进行各种分析.对于红细胞来说,它的裂解产物也称“溶血液”,其中主要成分有血红蛋白A1,A2,A3和碳酸酐酶(CA)等.本实验室用未裂解的完整的活体红细胞直接进行电泳,观察其释放出来的血红蛋白(hemoglobin,Hb),建立了淀粉-琼脂糖混合凝胶中红细胞的电泳释放实验.电泳释放可分为“初释放”(一次通电完成电泳,此时有Hb释放出来)和“再释放”(电泳过程中断电-再通电,又有Hb释放出来).本实验室在“初释放”实验中发现了“HbA2现象”,并通过Hb交叉电泳发现了HbA2与HbA1的相互作用;利用初释放型双向对角线电泳发现红细胞内HbA2与HbA1结合存在;对电泳释放出来的“HbA2现象”成分做SDS-PAGE及质谱分析,发现Prx-2(Peroxiredoxin-2)可能参与“HbA2现象”的形成;在研究“再释放”实验中发现了“Hb多带再释放现象”,在此基础上创建等渗再释放、低渗再释放、等低渗全程再释放及再释放型双向对角线电泳;两种红细胞(全血中的红细胞和由它分离出来的游离红细胞)再释放的比较研究;血浆成分对红细胞再释放的影响等.以上研究方法的建立为活体细胞内蛋白质存在状态的研究提供了基础,并开辟了新的研究途径和领域.%Nowadays, proteomics research is almost always made by lysing cells to release protein first and then to analyze the protein solution in many ways, the lytic product of red blood cells is also called hemolysate, the main components of which are hemoglobin A1, A2, A3 and carbonic anhydrase (CA), etc. In our laboratory, intact (unlysed) living red blood cells are used for direct electrophoresis so as to observe the released hemoglobin (Hb). Thus an electophoresis release experiment has been established with starch-agarose mixed gel. Electrophoresis release can be classified as

  4. DNA-SMART: Biopatterned Polymer Film Microchannels for Selective Immobilization of Proteins and Cells.

    Science.gov (United States)

    Schneider, Ann-Kathrin; Nikolov, Pavel M; Giselbrecht, Stefan; Niemeyer, Christof M

    2017-02-22

    A novel SMART module, dubbed "DNA-SMART" (DNA substrate modification and replication by thermoforming) is reported, where polymer films are premodified with single-stranded DNA capture strands, microthermoformed into 3D structures, and postmodified with complementary DNA-protein conjugates to realize complex biologically active surfaces within microfluidic devices. As a proof of feasibility, it is demonstrated that microchannels presenting three different proteins on their inner curvilinear surface can be used for selective capture of cells under flow conditions.

  5. Chemical surface modification of parylene C for enhanced protein immobilization and cell proliferation.

    Science.gov (United States)

    Zhang, Changhong; Thompson, Mark E; Markland, Frank S; Swenson, Steve

    2011-10-01

    To introduce the adhesion site of proteins and/or cells on parylene C (PC)-coated medical devices that can be used as implantable biosensors or drug delivery capsules, the PC surfaces were initially modified by the Friedel-Crafts acylation reaction to generate active chlorines. These chlorines were then employed to initiate the atom transfer radical polymerization of tert-butyl acrylate (TBA) and form a polymer brush layer of polyTBA on PC; the acrylate groups in the polymer brushes were hydrolyzed to carboxylic acid groups and further activated into succinimidyl ester groups via the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide coupling reaction. The PC surface grafted with polymer brushes and activated by succinimide showed efficient attachment of proteins, including gelatin, contortrostatin (CN) and bovine serum albumin (BSA), all at high density on the PC surface. The CN density on the surface was evaluated for both monolayer and polymer brush-based coatings. Based on fluorescence measurements, the polymer brush gives a 60-fold higher surface protein density than the monolayer-based system. Gelatin was used as a model protein and covalently coated onto the modified PC surface for cell culture study. Substrates with gelatin coating showed a significantly higher cell attachment and proliferation in 7 days cultures as compared to the uncoated substrates. In addition, a conventional photolithography technique was coupled with the surface chemistry to successfully pattern the BSA labeled with fluorescein isothiocyanate on the modified PC surfaces.

  6. Generation of continuous packed bed reactor with PVA-alginate blend immobilized Ochrobactrum sp. DGVK1 cells for effective removal of N,N-dimethylformamide from industrial effluents

    Energy Technology Data Exchange (ETDEWEB)

    Sanjeev Kumar, S.; Kumar, M. Santosh [Department of Biochemistry, Gulbarga University, Gulbarga 585106, Karnataka (India); Siddavattam, D. [Department of Animal Sciences, University of Hyderabad, Hyderabad 500046 (India); Karegoudar, T.B., E-mail: goudartbk@gmail.com [Department of Biochemistry, Gulbarga University, Gulbarga 585106, Karnataka (India)

    2012-01-15

    Highlights: Black-Right-Pointing-Pointer Removal of DMF was compared by free and immobilized cells of Ochrobactrum sp. DGVK1. Black-Right-Pointing-Pointer Ochrobactrum sp. DGVK1 cells entrapped in PVA-alginate have shown more tolerance. Black-Right-Pointing-Pointer PVA-alginate beads removed DMF even in the presence of other organic solvents. Black-Right-Pointing-Pointer Removal of DMF from industrial effluents by PVA-alginate blended batch operations. Black-Right-Pointing-Pointer Development of industrially feasible remediation strategy for DMF removal. - Abstract: Effective removal of dimethylformamide (DMF), the organic solvent found in industrial effluents of textile and pharma industries, was demonstrated by using free and immobilized cells of Ochrobactrum sp. DGVK1, a soil isolate capable of utilizing DMF as a sole source of carbon, nitrogen. The free cells have efficiently removed DMF from culture media and effluents, only when DMF concentration was less than 1% (v/v). Entrapment of cells either in alginate or in polyvinyl alcohol (PVA) failed to increase tolerance limits. However, the cells of Ochrobactrum sp. DGVK1 entrapped in PVA-alginate mixed matrix tolerated higher concentration of DMF (2.5%, v/v) and effectively removed DMF from industrial effluents. As determined through batch fermentation, these immobilized cells have retained viability and degradability for more than 20 cycles. A continuous packed bed reactor, generated by using PVA-alginate beads, efficiently removed DMF from industrial effluents, even in the presence of certain organic solvents frequently found in effluents along with DMF.

  7. Silk fibroin immobilization on poly(ethylene terephthalate) films: comparison of two surface modification methods and their effect on mesenchymal stem cells culture.

    Science.gov (United States)

    Liang, Meini; Yao, Jinrong; Chen, Xin; Huang, Lei; Shao, Zhengzhong

    2013-04-01

    Silk fibroin (SF) has played a curial role for the surface modification of conventional materials to improve the biocompatibility, and SF modified poly(ethylene terephthalate) (PET) materials have potential applications on tissue engineering such as artificial ligament, artificial vessel, artificial heart valve sewing cuffs dacron and surgical mesh engineering. In this work, SF was immobilized onto PET film via two different methods: 1) plasma pretreatment followed by SF dip coating (PET-SF) and 2) plasma-induce acrylic acid graft polymerization and subsequent covalent immobilization of SF on PET film (PET-PAA-SF). It could be found that plasma treatment provided higher surface roughness which was suitable for further SF dip coating, while grafted poly(acrylic acid) (PAA) promised the covalent bonding between SF and PAA. ATR-FTIR adsorption band at 3284 cm(-1), 1623 cm(-1) and 1520 cm(-1) suggested the successful introduction of SF onto PET surface, while the amount of immobilized SF of PET-SF was higher than PET-PAA-SF according to XPS investigation (0.29 vs 0.23 for N/C ratio). Surface modified PET film was used as substrate for mesenchymal stem cells (MSCs) culture, the cells on PET-SF surface exhibited optimum density compared to PET-PAA-SF according to CCK-8 assays, which indicated that plasma pretreatment followed by SF dip coating was a simple and effective way to prepare biocompatible PET surface.

  8. Arginine-assisted immobilization of silver nanoparticles on ZnO nanorods: an enhanced and reusable antibacterial substrate without human cell cytotoxicity

    Science.gov (United States)

    Agnihotri, Shekhar; Bajaj, Geetika; Mukherji, Suparna; Mukherji, Soumyo

    2015-04-01

    Silver-based hybrid nanomaterials are gaining interest as potential alternatives for conventional antimicrobial agents. Herein, we present a simple, facile and eco-friendly approach for the deposition of silver nanoparticles (AgNPs) on ZnO nanorods, which act as a nanoreactor for in situ synthesis and as an immobilizing template in the presence of arginine. The presence of arginine enhanced the stability of ZnO deposition on the glass substrate by hindering the dissolution of zinc under alkaline conditions. Various Ag/ZnO hybrid nanorod (HNR) samples were screened to obtain a high amount of silver immobilization on the ZnO substrate. Ag/ZnO HNRs displayed potent antibacterial ability and could achieve 100% kill for both Escherichia coli and Bacillus subtilis strains under various test conditions. The hybrid material mediated its dual mode of antibacterial action through direct contact-killing and release of silver ions/nanoparticles and showed superior bactericidal performance compared to pure ZnO nanorods and colloidal AgNPs. No significant decline in antibacterial efficacy was observed even after the same substrate was repeatedly reused multiple times. Interestingly, the amount of Ag and Zn release was much below their maximal limit in drinking water, thus preventing potential health hazards. Immobilized AgNPs showed no cytotoxic effects on the human hepatocarcinoma cell line (HepG2). Moreover, treating cells with the antibacterial substrate for 24 hours did not lead to significant generation of reactive oxygen species (ROS). The good biocompatibility and bactericidal efficacy would thus make it feasible to utilize this immobilization strategy for preparing new-generation antibacterial coatings.Silver-based hybrid nanomaterials are gaining interest as potential alternatives for conventional antimicrobial agents. Herein, we present a simple, facile and eco-friendly approach for the deposition of silver nanoparticles (AgNPs) on ZnO nanorods, which act as a

  9. Shaping Crystals using Electrophoresis

    Science.gov (United States)

    Palacci, Jeremie; Mackiewicz, Kristian

    2016-11-01

    Electrophoresis is size and shape independent as stressed by Morrison in his seminal paper. Here we present an original approach to reshape colloidal crystals using an electric field as a carving tool.

  10. Electrophoresis operations in space

    Science.gov (United States)

    Richman, D. W.

    1982-01-01

    Application of electrophoresis in space processing is described. Spaceborne experiments in areas such as biological products and FDA approved drugs are discussed. These experiments will be carried on shuttle payloads.

  11. Protein electrophoresis - serum

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003540.htm Protein electrophoresis - serum To use the sharing features on ... JavaScript. This lab test measures the types of protein in the fluid (serum) part of a blood ...

  12. Electrophoresis in strong electric fields.

    Science.gov (United States)

    Barany, Sandor

    2009-01-01

    Two kinds of non-linear electrophoresis (ef) that can be detected in strong electric fields (several hundred V/cm) are considered. The first ("classical" non-linear ef) is due to the interaction of the outer field with field-induced ionic charges in the electric double layer (EDL) under conditions, when field-induced variations of electrolyte concentration remain to be small comparatively to its equilibrium value. According to the Shilov theory, the non-linear component of the electrophoretic velocity for dielectric particles is proportional to the cubic power of the applied field strength (cubic electrophoresis) and to the second power of the particles radius; it is independent of the zeta-potential but is determined by the surface conductivity of particles. The second one, the so-called "superfast electrophoresis" is connected with the interaction of a strong outer field with a secondary diffuse layer of counterions (space charge) that is induced outside the primary (classical) diffuse EDL by the external field itself because of concentration polarization. The Dukhin-Mishchuk theory of "superfast electrophoresis" predicts quadratic dependence of the electrophoretic velocity of unipolar (ionically or electronically) conducting particles on the external field gradient and linear dependence on the particle's size in strong electric fields. These are in sharp contrast to the laws of classical electrophoresis (no dependence of V(ef) on the particle's size and linear dependence on the electric field gradient). A new method to measure the ef velocity of particles in strong electric fields is developed that is based on separation of the effects of sedimentation and electrophoresis using videoimaging and a new flowcell and use of short electric pulses. To test the "classical" non-linear electrophoresis, we have measured the ef velocity of non-conducting polystyrene, aluminium-oxide and (semiconductor) graphite particles as well as Saccharomice cerevisiae yeast cells as a

  13. Arginine-assisted immobilization of silver nanoparticles on ZnO nanorods: an enhanced and reusable antibacterial substrate without human cell cytotoxicity.

    Science.gov (United States)

    Agnihotri, Shekhar; Bajaj, Geetika; Mukherji, Suparna; Mukherji, Soumyo

    2015-04-28

    Silver-based hybrid nanomaterials are gaining interest as potential alternatives for conventional antimicrobial agents. Herein, we present a simple, facile and eco-friendly approach for the deposition of silver nanoparticles (AgNPs) on ZnO nanorods, which act as a nanoreactor for in situ synthesis and as an immobilizing template in the presence of arginine. The presence of arginine enhanced the stability of ZnO deposition on the glass substrate by hindering the dissolution of zinc under alkaline conditions. Various Ag/ZnO hybrid nanorod (HNR) samples were screened to obtain a high amount of silver immobilization on the ZnO substrate. Ag/ZnO HNRs displayed potent antibacterial ability and could achieve 100% kill for both Escherichia coli and Bacillus subtilis strains under various test conditions. The hybrid material mediated its dual mode of antibacterial action through direct contact-killing and release of silver ions/nanoparticles and showed superior bactericidal performance compared to pure ZnO nanorods and colloidal AgNPs. No significant decline in antibacterial efficacy was observed even after the same substrate was repeatedly reused multiple times. Interestingly, the amount of Ag and Zn release was much below their maximal limit in drinking water, thus preventing potential health hazards. Immobilized AgNPs showed no cytotoxic effects on the human hepatocarcinoma cell line (HepG2). Moreover, treating cells with the antibacterial substrate for 24 hours did not lead to significant generation of reactive oxygen species (ROS). The good biocompatibility and bactericidal efficacy would thus make it feasible to utilize this immobilization strategy for preparing new-generation antibacterial coatings.

  14. Moving into advanced nanomaterials. Toxicity of rutile TiO2 nanoparticles immobilized in nanokaolin nanocomposites on HepG2 cell line.

    Science.gov (United States)

    Bessa, Maria João; Costa, Carla; Reinosa, Julian; Pereira, Cristiana; Fraga, Sónia; Fernández, José; Bañares, Miguel A; Teixeira, João Paulo

    2017-02-01

    Immobilization of nanoparticles on inorganic supports has been recently developed, resulting in the creation of nanocomposites. Concerning titanium dioxide nanoparticles (TiO2 NPs(1)), these have already been developed in conjugation with clays, but so far there are no available toxicological studies on these nanocomposites. The present work intended to evaluate the hepatic toxicity of nanocomposites (C-TiO2(2)), constituted by rutile TiO2 NPs immobilized in nanokaolin (NK(3)) clay, and its individual components. These nanomaterials were analysed by means of FE-SEM(4) and DLS(5) analysis for physicochemical characterization. HepG2 cells were exposed to rutile TiO2 NPs, NK clay and C-TiO2 nanocomposite, in the presence and absence of serum for different exposure periods. Possible interferences with the methodological procedures were determined for MTT,(6) neutral red uptake, alamar blue (AB), LDH,(7) and comet assays, for all studied nanomaterials. Results showed that MTT, AB and alkaline comet assay were suitable for toxicity analysis of the present materials after slight modifications to the protocol. Significant decreases in cell viability were observed after exposure to all studied nanomaterials. Furthermore, an increase in HepG2 DNA damage was observed after shorter periods of exposure in the absence of serum proteins and longer periods of exposure in their presence. Although the immobilization of nanoparticles in micron-sized supports could, in theory, decrease the toxicity of single nanoparticles, the selection of a suitable support is essential. The present results suggest that NK clay is not the appropriate substrate to decrease TiO2 NPs toxicity. Therefore, for future studies, it is critical to select a more appropriate substrate for the immobilization of TiO2 NPs.

  15. Tomography of a Cryo-immobilized Yeast Cell Using Ptychographic Coherent X-Ray Diffractive Imaging.

    Science.gov (United States)

    Giewekemeyer, K; Hackenberg, C; Aquila, A; Wilke, R N; Groves, M R; Jordanova, R; Lamzin, V S; Borchers, G; Saksl, K; Zozulya, A V; Sprung, M; Mancuso, A P

    2015-11-03

    The structural investigation of noncrystalline, soft biological matter using x-rays is of rapidly increasing interest. Large-scale x-ray sources, such as synchrotrons and x-ray free electron lasers, are becoming ever brighter and make the study of such weakly scattering materials more feasible. Variants of coherent diffractive imaging (CDI) are particularly attractive, as the absence of an objective lens between sample and detector ensures that no x-ray photons scattered by a sample are lost in a limited-efficiency imaging system. Furthermore, the reconstructed complex image contains quantitative density information, most directly accessible through its phase, which is proportional to the projected electron density of the sample. If applied in three dimensions, CDI can thus recover the sample's electron density distribution. As the extension to three dimensions is accompanied by a considerable dose applied to the sample, cryogenic cooling is necessary to optimize the structural preservation of a unique sample in the beam. This, however, imposes considerable technical challenges on the experimental realization. Here, we show a route toward the solution of these challenges using ptychographic CDI (PCDI), a scanning variant of coherent imaging. We present an experimental demonstration of the combination of three-dimensional structure determination through PCDI with a cryogenically cooled biological sample--a budding yeast cell (Saccharomyces cerevisiae)--using hard (7.9 keV) synchrotron x-rays. This proof-of-principle demonstration in particular illustrates the potential of PCDI for highly sensitive, quantitative three-dimensional density determination of cryogenically cooled, hydrated, and unstained biological matter and paves the way to future studies of unique, nonreproducible biological cells at higher resolution.

  16. Efficient anti-Prelog enantioselective reduction of acetyltrimethylsilane to (R-1-trimethylsilylethanol by immobilized Candida parapsilosis CCTCC M203011 cells in ionic liquid-based biphasic systems

    Directory of Open Access Journals (Sweden)

    Zhang Bo-Bo

    2012-08-01

    Full Text Available Abstract Background Biocatalytic asymmetric reductions with whole cells can offer high enantioselectivity, environmentally benign processes and energy-effective operations and thus are of great interest. The application of whole cell-mediated bioreduction is often restricted if substrate and product have low water solubility and/or high toxicity to the biocatalyst. Many studies have shown that a biphasic system is often useful in this instance. Hence, we developed efficient biphasic reaction systems with biocompatible water-immiscible ionic liquids (ILs, to improve the biocatalytic anti-Prelog enantioselective reduction of acetyltrimethylsilane (ATMS to (R-1-trimethylsilylethanol {(R-1-TMSE}, which is key synthon for a large number of silicon-containing drugs, using immobilized Candida parapsilosis CCTCC M203011 cells as the biocatalyst. Results It was found that the substrate ATMS and the product 1-TMSE exerted pronounced toxicity to immobilized Candida parapsilosis CCTCC M203011 cells. The biocompatible water-immiscible ILs can be applied as a substrate reservoir and in situ extractant for the product, thus greatly enhancing the efficiency of the biocatalytic process and the operational stability of the cells as compared to the IL-free aqueous system. Various ILs exerted significant but different effects on the bioreduction and the performances of biocatalysts were closely related to the kinds and combination of cation and anion of ILs. Among all the water-immiscible ILs investigated, the best results were observed in 1-butyl-3-methylimidazolium hexafluorophosphate (C4mim·PF6/buffer biphasic system. Furthermore, it was shown that the optimum substrate concentration, volume ratio of buffer to IL, buffer pH, reaction temperature and shaking rate for the bioreduction were 120 mM, 8/1 (v/v, 6.0, 30°C and 180 r/min, respectively. Under these optimized conditions, the initial reaction rate, the maximum yield and the product e.e. were 8.1

  17. Recent advances in preparative electrophoresis

    Science.gov (United States)

    Mosher, Richard A.; Thormann, Wolfgang; Egen, Ned B.; Couasnon, Pascal; Sammons, David W.

    1987-01-01

    Various approaches for preparative electrophoresis, and three new instruments for preparative electrophoresis are discussed. Consideration is given to isoelectric focusing, isotachophoresis, and zone electrophoresis, three gel-based electrophoresis methods. The design, functions, and performance of the Elphor VaP 21 device of Hannig (1982), the shear-stabilized BIOSTREAM separator of Thompson (1983), and the recycling isoelectric focusing device are described.

  18. The application of methylation specific electrophoresis (MSE to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

    Directory of Open Access Journals (Sweden)

    Yokoyama Seiya

    2012-02-01

    Full Text Available Abstract Background Methylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity. Methods Here, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE. The MSE method is comprised of the following steps: (a bisulfite treatment of genomic DNA, (b amplification of the target DNA by a nested PCR approach and (c applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices. Result The MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is Conclusions The MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical samples, and this may facilitate identification of new risk markers.

  19. Electrophoresis of diffuse soft particles.

    Science.gov (United States)

    Duval, Jérôme F L; Ohshima, Hiroyuki

    2006-04-11

    A theory is presented for the electrophoresis of diffuse soft particles in a steady dc electric field. The particles investigated consist of an uncharged impenetrable core and a charged diffuse polyelectrolytic shell, which is to some extent permeable to ions and solvent molecules. The diffuse character of the shell is defined by a gradual distribution of the density of polymer segments in the interspatial region separating the core from the bulk electrolyte solution. The hydrodynamic impact of the polymer chains on the electrophoretic motion of the particle is accounted for by a distribution of Stokes resistance centers. The numerical treatment of the electrostatics includes the possibility of partial dissociation of the hydrodynamically immobile ionogenic groups distributed throughout the shell as well as specific interaction between those sites with ions from the background electrolyte other than charge-determining ions. Electrophoretic mobilities are computed on the basis of an original numerical scheme allowing rigorous evaluation of the governing transport and electrostatic equations derived following the strategy reported by Ohshima, albeit within the restricted context of a discontinuous chain distribution. Attention is particularly paid to the influence of the type of distribution adopted on the electrophoretic mobility of the particle as a function of its size, charge, degree of permeability, and solution composition. The results are systematically compared with those obtained with a discontinuous representation of the interface. The theory constitutes a basis for interpreting electrophoretic mobilities of heterogeneous systems such as environmental or biological colloids or swollen/deswollen microgel particles.

  20. Detection of DNA damage by alkaline single cell gel electrophoresis in 2,4-dichlorophenoxyacetic-acid- and butachlor-exposed erythrocytes of Clarias batrachus.

    Science.gov (United States)

    Ateeq, Bushra; Abul Farah, M; Ahmad, Waseem

    2005-11-01

    The alkaline single cell gel electrophoresis, also known as comet assay, is a rapid, simple and sensitive technique for measuring DNA strand breaks in individual cells. The present study was undertaken to evaluate the genotoxic potential of two widely used herbicides; 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-chloro-2,6-diethyl-N-(butoxymethyl) acetanilide (butachlor) in erythrocytes of freshwater catfish, Clarias batrachus. Fish were exposed by medium treatment with three sub-lethal concentrations of 2,4-D (25, 50, and 75ppm) and butachlor (1, 2, and 2.5ppm) and alkaline comet assay was performed on nucleated erythrocytes after 48, 72, and 96h. The amount of DNA damage in cells was estimated from comet tail length as the extent of migration of the genetic material. A significant increase in comet tail length indicating DNA damage was observed at all concentrations of both the herbicides compared with control (Pbutachlor (9.28microm). This study confirmed that the comet assay applied on the fish erythrocyte is a useful tool in determining potential genotoxicity of water pollutants and might be appropriate as a part of a monitoring program.

  1. Genotoxicity assessment of antidiabetic formulation (ADPHF6 in human lymphocytes by single cell gel electrophoresis (comet assay - an in vitro study

    Directory of Open Access Journals (Sweden)

    Devanand Shanmugasundaram

    2015-06-01

    Full Text Available Levels of Reactive Oxygen Species (ROS molecules during aerobic metabolism are often regulated by unique endogenous antioxidant system. During hyperglycaemic condition, accumulation of excess fatty acids & glucose in adipose tissue (Wright Jr E., 2006 results in increased levels of ROS. When ROS molecules overwhelms the cells antioxidant defence system, it ends up in cellular oxidative stress; which in turn is reported to cause oxidative DNA damage & intervene damage to macromolecules & cellular membranes (Ahmad et al., 2013. Our novel anti-hyperglycaemic polyherbal formulation (ADPHF6 had already illustrated significant inhibitory activity against α-amylase & α-glucosidase enzymes and also scavenging free radicals (in vitro models. The present study demonstrates the protective effect of formulation against H2O2 induced DNA damage in human lymphocytes by Single Cell Gel Electrophoresis (SCGE assay. Experimental procedures were approved by Institutional Human Ethics Committee of Frontier Lifeline Hospital, Chennai, India (FLL/IEC/02/2014. Peripheral human lymphocytes were isolated (Duthie et.al, 2002 and subjected for Cell viability by Trypan blue exclusion method. The alkaline SCGE assay was carried out to determine the level of DNA damage in ADPHF6 treated cells with minor modifications from Singh et al., 1988. Frosted microscopic slides were pre-coated with 1% NMA followed by 1% LMA and incubated for 15 min at 15-20o C. 100 μL of freshly prepared cell suspension (2 x 104 cells was mixed with 0.5% LMA & casted on microscopic slide. The cells were immersed in lysing solution for 2 hours at 4O C and washed in TBE buffer for 5 min at RT. All the slides were treated with fresh alkaline solution for 20 minutes for expression of alkali-labile damage. Electrophoresis was performed at 24 V for 20 min at RT. Slides were washed in neutralizing buffer for 5 min at RT. All the groups were stained with Acridine Orange (20µg/ml & Propidium Iodide (20µg

  2. The combined effects of matrix stiffness and growth factor immobilization on the bioactivity and differentiation capabilities of adipose-derived stem cells.

    Science.gov (United States)

    Banks, Jessica M; Mozdzen, Laura C; Harley, Brendan A C; Bailey, Ryan C

    2014-10-01

    Biomaterial designs are increasingly incorporating multiple instructive signals to induce a desired cell response. However, many approaches do not allow orthogonal manipulation of immobilized growth factor signals and matrix stiffness. Further, few methods support patterning of biomolecular signals across a biomaterial in a spatially-selective manner. Here, we report a sequential approach employing carbodiimide crosslinking and benzophenone photoimmobilization chemistries to orthogonally modify the stiffness and immobilized growth factor content of a model collagen-GAG (CG) biomaterial. We subsequently examined the singular and combined effects of bone morphogenetic protein (BMP-2), platelet derived growth factor (PDGF-BB), and CG membrane stiffness on the bioactivity and osteogenic/adipogenic lineage-specific gene expression of adipose derived stem cells, an increasingly popular cell source for regenerative medicine studies. We found that the stiffest substrates direct osteogenic lineage commitment of ASCs regardless of the presence or absence of growth factors, while softer substrates require biochemical cues to direct cell fate. We subsequently describe the use of this approach to create overlapping patterns of growth factors across a single substrate. These results highlight the need for versatile approaches to selectively manipulate the biomaterial microenvironment to identify synergies between biochemical and mechanical cues for a range of regenerative medicine applications.

  3. Determination of sulfur and selected trace elements in metallothionein-like proteins using capillary electrophoresis hyphenated to inductively coupled plasma mass spectrometry with an octopole reaction cell

    Energy Technology Data Exchange (ETDEWEB)

    Proefrock, Daniel; Leonhard, Peter; Prange, Andreas [GKSS Research Centre Geesthacht, Institute for Coastal Research, Max Planck Strasse, 21502, Geesthacht (Germany)

    2003-09-01

    The determination of sulfur in biologically relevant samples such as metalloproteins is described. The analytical methodology used is based on robust on-line coupling between capillary electrophoresis (CE) and octopole reaction cell inductively-coupled plasma mass spectrometry (ORC-ICP-MS). Polyatomic ions that form in the plasma and interfere with the determination of S at mass 32 are minimised by addition of xenon to the collision cell. The method has been applied to the separation and simultaneous element-specific detection of sulfur, cadmium, copper, and zinc in commercially available metallothionein preparations (MT) and metallothionein-like proteins (MLP) extracted from liver samples of bream (Abramis brama L.) caught in the river Elbe, Germany. Instrumental detection limits have been calculated according to the German standard procedure DIN 32645 for the determination of sulfur and some simultaneously measured trace elements in aqueous solution. For sulfur detection limits down to 1.3 {mu}g L{sup -1} ({sup 34}S) and 3.2 {mu}g L{sup -1} ({sup 32}S) were derived. For the other trace elements determined simultaneously detection limits ranging from 300 ng L{sup -1} ({sup 58}Ni) to 500 ng L{sup -1} ({sup 66}Zn, {sup 55}Mn) were achieved. For quantification of sulfur and cadmium in a commercially available MT preparation under hyphenated conditions the use of external calibration is suggested. Finally, the need for proper sample-preparation technique will be discussed. (orig.)

  4. Bioconversion of l-glutamic acid to α-ketoglutaric acid by an immobilized whole-cell biocatalyst expressing l-amino acid deaminase from Proteus mirabilis.

    Science.gov (United States)

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

    2014-01-01

    The goal of this work was to develop an immobilized whole-cell biocatalytic process for the environment-friendly synthesis of α-ketoglutaric acid (α-KG) from l-glutamic acid. We compared the suitability of Escherichia coli and Bacillus subtilis strains overexpressing Proteus mirabilisl-amino acid deaminase (l-AAD) as potential biocatalysts. Although both recombinant strains were biocatalytically active, the performance of B. subtilis was superior to that of E. coli. With l-glutamic acid as the substrate, α-KG production levels by membranes isolated from B. subtilis and E. coli were 55.3±1.73 and 21.7±0.39μg/mg protein/min, respectively. The maximal conversion ratio of l-glutamic acid to α-KG was 31% (w/w) under the following optimal conditions: 15g/L l-glutamic acid, 20g/L whole-cell biocatalyst, 5mM MgCl2, 40°C, pH 8.0, and 24-h incubation. Immobilization of whole cells with alginate increased the recyclability by an average of 23.33% per cycle. This work established an efficient one-step biotransformation process for the production of α-KG using immobilized whole B. subtilis overexpressing P. mirabilisl-AAD. Compared with traditional multistep chemical synthesis, the biocatalytic process described here has the advantage of reducing environmental pollution and thus has great potential for the large-scale production of α-KG.

  5. Assessment of the red cell proteome of young patients with unexplained hemolytic anemia by two-dimensional differential in-gel electrophoresis (DIGE.

    Directory of Open Access Journals (Sweden)

    Katharina von Löhneysen

    Full Text Available Erythrocyte cytosolic protein expression profiles of children with unexplained hemolytic anemia were compared with profiles of close relatives and controls by two-dimensional differential in-gel electrophoresis (2D-DIGE. The severity of anemia in the patients varied from compensated (i.e., no medical intervention required to chronic transfusion dependence. Common characteristics of all patients included chronic elevation of reticulocyte count and a negative workup for anemia focusing on hemoglobinopathies, morphologic abnormalities that would suggest a membrane defect, immune-mediated red cell destruction, and evaluation of the most common red cell enzyme defects, glucose-6-phosphate dehydrogenase and pyruvate kinase deficiency. Based upon this initial workup and presentation during infancy or early childhood, four patients classified as hereditary nonspherocytic hemolytic anemia (HNSHA of unknown etiology were selected for proteomic analysis. DIGE analysis of red cell cytosolic proteins clearly discriminated each anemic patient from both familial and unrelated controls, revealing both patient-specific and shared patterns of differential protein expression. Changes in expression pattern shared among the four patients were identified in several protein classes including chaperons, cytoskeletal and proteasome proteins. Elevated expression in patient samples of some proteins correlated with high reticulocyte count, likely identifying a subset of proteins that are normally lost during erythroid maturation, including proteins involved in mitochondrial metabolism and protein synthesis. Proteins identified with patient-specific decreased expression included components of the glutathione synthetic pathway, antioxidant pathways, and proteins involved in signal transduction and nucleotide metabolism. Among the more than 200 proteins identified in this study are 21 proteins not previously described as part of the erythrocyte proteome. These results

  6. 固定化细胞滤床和生物膜滤床净化二甲苯的比较%Comparison of air-borne xylene biodegradation between immobilized-cell biofilter and biofilm attached biofilter

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The Bacillus firmus was immobilized into Ca- alginate beads according to the different initial biomass concentration, calcification time and activation time. Three types of immobilized Bacillus firmus beads were packed respectively in trickling biofilter to purify xylene contained waste gases, and the performance of immobilized-cell biofilter was compared with traditional biofilm attached biofilter packed with two types of ceramic pellets. The results showed that three types of immobilized beads had different capabilities for removing xylene and life-spans. Higher initial biomass in immobilized beads resulted in better performance but shorter life-span. Activation process can remarkably enhance the activity of bacteria, and the removal efficiency of xylene can substantially be improved. Calcification time had influence on life-span of immobilized beads. Without acclimation, the cell-entrapped biofilter can obtain the maximum elimination capacity of 92.4 g/(m3·h). However, compared with biofilm attached biofilter, it has a poorer intrinsic drawback in volatile organic compounds (VOCs) removal due to the existence of excess mass transfer resistance.

  7. Determination of glutathione in single HepG2 cells by capillary electrophoresis with reduced graphene oxide modified microelectrode.

    Science.gov (United States)

    Wang, Xiaolei; Wang, Jun; Fu, Hongyan; Liu, Dongju; Chen, Zhenzhen

    2014-12-01

    Determination of intracellular bioactive species will afford beneficial information related to cell metabolism, signal transduction, cell function, and disease treatment. In this study, the electrochemically reduced graphene oxide modified carbon fiber microdisk electrode (ER-GOME) was used as a detector of CZE-electrochemical detection and developed to detect glutathione (GSH). The electrocatalytic activity of the modified microelectrode was characterized by cyclic voltammetry. Under optimized experimental conditions, the concentration linear range of GSH was from 1 to 60 μM. When the S/N ratio was 3, the concentration detection limit was 1 μM. Compared with the unmodified carbon fiber microdisk electrode, the sensitivity was enhanced more than five times. With the use of this method, the average contents of GSH in single HepG2 cells were found to be 7.13 ± 1.11 fmol (n = 10). Compared with gold/mercury amalgam microelectrode, which was usually used in determining GSH, the electrochemically reduced graphene oxide modified carbon fiber microdisk electrode was friendly to environment for free mercury. Furthermore, there were several merits of the novel electrochemical detector coupled with CE, such as comparative repeatability, easy fabrication, and high sensitivity, hold great potential for the single-cell assay.

  8. Optimization of date syrup for enhancement of the production of citric acid using immobilized cells of Aspergillus niger

    OpenAIRE

    Mostafa, Yasser S.; Alamri, Saad A.

    2012-01-01

    Date syrup as an economical source of carbohydrates and immobilized Aspergillus niger J4, which was entrapped in calcium alginate pellets, were employed for enhancing the production of citric acid. Maximum production was achieved by pre-treating date syrup with 1.5% tricalcium phosphate to remove heavy metals. The production of citric acid using a pretreated medium was 38.87% higher than an untreated one that consumed sugar. The appropriate presence of nitrogen, phosphate and magnesium appear...

  9. DNA ELECTROPHORESIS AT SURFACES

    Energy Technology Data Exchange (ETDEWEB)

    RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

    2003-09-01

    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  10. Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection

    Directory of Open Access Journals (Sweden)

    Yang Wang

    2013-03-01

    Full Text Available The N-linked glycosylation in recombinant monoclonal antibodies (mAb occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC and complement-dependent cytotoxicity (CDC through their interaction with Fc-receptors. Hence, it is critical to monitor and characterize the N-linked glycosylation profile in a therapeutic protein such as a mAb for product consistency. In one approach, the glycans are first released from the mAb using an enzyme specific digestion, such as Protein N-Glycosidase F (PNGase and subsequently they are labeled using a fluorophore, for example, 8-aminopyrene-1,3,6-trisulfonic acid (APTS . Here we have applied this approach and used Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF to analyze a recombinant mAb produced in murine myeloma (NS0 cells. The technique provides short analysis times, efficient separations, and high sensitivity. CE-LIF peak identification was done by a combination of glycan standards and treatment with various exoglycosidases. Furthermore, the APTS-labeled glycans were also analyzed using hydrophilic interaction chromatography (HILIC high performance liquid chromatography (HPLC to aid identification of minor peaks by sample collection and off-line mass spectrometry (MS analysis.

  11. Detection of adenosine triphosphate in HeLa cell using capillary electrophoresis-laser induced fluorescence detection based on aptamer and graphene oxide.

    Science.gov (United States)

    Fang, Bi-Yun; Yao, Ming-Hao; Wang, Chun-Yuan; Wang, Chao-Yang; Zhao, Yuan-Di; Chen, Fang

    2016-04-01

    A method for ATP quantification based on dye-labeled aptamer/graphene oxide (aptamer/GO) using capillary electrophoresis-laser induced fluorescence (CE-LIF) detecting technique has been established. In this method, the carboxyfluorescein (FAM)-labelled ATP aptamers were adsorbed onto the surface of GO, leading to the fluorescence quenching of FAM; after the incubation with a limited amount of ATP, stronger affinity between ATP aptamer and ATP resulted in the desorption of aptamers and the fluorescence restoration of FAM. Then, aptamer-ATP complex and excess of aptamer/GO and GO were separated and quantified by CE-LIF detection. It was shown that a linear relation was existing in the CE-LIF peak intensity of aptamer-ATP and ATP concentration in range of 10-700 μM, the regression equation was F=1.50+0.0470C(ATP) (R(2)=0.990), and the limit of detection was 1.28 μM (3S/N, n=5), which was one order magnitude lower than that of detection in solution by fluorescence method. The approach with excellent specificity and reproducibility has been successfully applied to detecting concentration of ATP in HeLa cell.

  12. In vivo genotoxicity of ortho-phenylphenol, biphenyl, and thiabendazole detected in multiple mouse organs by the alkaline single cell gel electrophoresis assay.

    Science.gov (United States)

    Sasaki, Y F; Saga, A; Akasaka, M; Yoshida, K; Nishidate, E; Su, Y Q; Matsusaka, N; Tsuda, S

    1997-12-12

    In Japan, ortho-phenylphenol (OPP), biphenyl (BP), and thiabendazole (2-(4'-thiazolyl)benzimidazole, TBZ) are commonly used as a postharvest treatment to preserve imported citrus fruits during transport and storage. We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of those agents in mouse stomach, liver, kidney, bladder, lung, brain, and bone marrow. CD-1 male mice were sacrificed 3, 8, and 24 h after oral administration of the test compounds. OPP (2000 mg/kg) induced DNA damage in the stomach, liver, kidney, bladder, and lung, BP (2000 mg/kg) and TBZ (200 mg/kg) induced DNA damage in all the organs studied. For OPP, increased DNA damage peaked at 3-8 h and tended to decrease at 24 h. For BP, on the contrary, increased DNA migration peaked at 24 h. That delay may have been due to the fact that OPP is metabolized by cytochrome 450 and prostaglandin H synthase to phenylbenzoquinone (PBQ), a DNA binding metabolite, and BP is metabolized to PBQ via OPP and m-phenylphenol. The positive response to TBZ, an aneugen, supports the in vivo DNA-damaging action of TBZ.

  13. Use of two-dimensional fluorescence spectroscopy for monitoring of the effect of dimethyl sulfoxide on the growth and viability of immobilized plant cells

    Directory of Open Access Journals (Sweden)

    Vankova Radomira

    2003-01-01

    Full Text Available The growth and viability of tobacco cells (Nicotiana tabacum L immobilized in alginate or pectate were monitored during their cultivation by using two-dimensional fluorescence spectroscopy (2-D FS. The cell growth was followed via the fluorescence of amino acids in proteins. The correlation between the tryptophan fluorescence and the cell biomass inside the alginate beads was verified by comparison with the dry weight of the cells. The determination of biomass content or cell viability by measurement of the intensity of NAD(PH fluorescence was found unsuitable. Cell viability was estimated by determination of cell esterase activity using fluorescein diacetate as a fluorogenic substrate. The fluorescence intensities of both fluorophores, tryptophan and fluorescein, were determined by scanning a 2-D FS spectrum of intact beads in front face cuvette. Using this technique the effect of organic solvent, dimethyl sulfoxide, on the growth and metabolic activities of cells within the beads was evaluated. While 4% DMSO was tolerated by cells, 6% DMSO led to the cell destruction.

  14. Fermentation of wood-derived acid hydrolysates in a batch bioreactor and in a continuous dynamic immobilized cell bioreactor by Pichia stipitis R

    Energy Technology Data Exchange (ETDEWEB)

    Parekh, S.R.; Parekh, R.S.; Wayman, M.

    1987-06-01

    Ethanolic fermentation of sugar solutions obtained on acid prehydrolysis of aspen and pine woods and from those present in spent sulphite liquor (SSL) were investigated in batch culture and in a continuous dynamic immobilized cell (CDIC) fermenter. The inhibitory effect of acetic acid on the fermentation of such liquors was overcome by steam-stripping. Major benefits were obtained when Pichia stipitis R was employed in fermenting and removing sugar from SSL compared to Saccharomyces cerevisiae. Ethanol yields were 38% higher along with 40% more sugar removal from softwood SSL. S. cerevisiae was not able to ferment hardwood SSL effectively. Using immobilized cells of P. stipitis R in a CDIC bioreactor, we found that softwood SSL could be fermented in seven hours with 90% xylose fermentation, while hemicellulose hydrolysates were fermented in four to five hours, with an average volumetric ethanol productivity of about 2.6 g/l/h. The merits of using P. stipitis R for efficient and rapid fermentation of xylose-rich hydrolysates, and the possible application of the CDIC bioreactor to continuous ethanolic fermentations are discussed. (Refs. 28).

  15. Response surface optimization for the transesterification of karanja oil using immobilized whole cells of Rhizopus oryzae in n-hexane system

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Devanesan; Rajendran, Aravindan; Thangavelu, Viruthagiri [Annamalai University, Department of Chemical Engineering, Faculty of Engineering and Technology, Biochemical Engineering Laboratory, Annamalai Nagar, Tamil Nadu (India)

    2012-03-15

    Non-edible oils represent one of the most viable alternative feed stocks for the production of large volumes of biodiesel at cheaper cost in tropical countries. The objective of the present study is to investigate the ability of the immobilized whole cells of Rhizopus oryzae MTCC 262 to catalyze the biodiesel production from karanja oil in n-hexane system. Response surface methodology was employed to evaluate the effects of synthesis parameters, such as molar ratio of oil to alcohol, reaction temperature and reaction time on percentage biodiesel (methyl esters) yield. Transesterification was performed in shake flasks containing immobilized cells in the reaction mixture with 10% oil weight of n-hexane. The quadratic effects of molar ratio of oil to alcohol and reaction time proved to be the significant at 1% and 5% levels, respectively. The optimum synthesis conditions were found to be: molar ratio of oil to alcohol 1:2.73, reaction temperature 41.39 C and reaction time 73.97 h. Biodiesel yield (methyl ester) was 75.98 (wt.%) under the optimal conditions and the subsequent verification experiments with biodiesel yield of 78.0 (wt.%) confirmed the validity of the proposed model. (orig.)

  16. Immobilization of nitrite oxidizing bacteria using biopolymeric chitosan media

    Institute of Scientific and Technical Information of China (English)

    Pranee Lertsutthiwong; Duangcheewan Boonpuak; Wiboonluk Pungrasmi; Sorawit Powtongsook

    2013-01-01

    The effects of chitosan characteristics including the degree of deacetylation,molecular weight,particle size,pH pretreatment and immobilization time on the immobilization of nitrite-oxidizing bacteria (NOB) on biopolymeric chitosan were investigated.Nitrite removal efficiency of immobilized NOB depended on the degree of deacetylation,particle size,pH pretreatment on the surface of chitosan and immobilization time.Scanning electron microscope characterization illustrated that the number of NOB cells attached to the surface of chitosan increased with an increment of immobilization time.The optimal condition for NOB immobilization on chitosan was achieved during a 24-hr immobilization period using chitosan with the degree of deacetylation larger than 80% and various particle size ranges between 1-5 mm at pH 6.5.In general,the NOB immobilized on chitosan flakes has a high potential to remove excess nitrite from wastewater and aquaculture systems.

  17. Pre-flight report on cultured human embryonic kidney cell handling and cell electrophoresis. Prepared prior to continuous-flow electrophoretic separation experiments aboard space shuttle flight STS-8

    Science.gov (United States)

    Todd, P. W.; Sarnoff, B. E.; Li, Z. K.

    1985-01-01

    Studies of the physical properties of continuous-flow zero-G electrophoretic separator (CFES) buffer, the electrokinetic properties of human erythrocytes in the CFES buffer, the electrokinetic properties of human embryonic kidney cells in the CFES buffer, and the viability and yield of human embryonc kidney cells subjected to flight handling procedures are discussed. In general, the procedure for cell handling and electrophoresis of HEK-8514 cells in 1st or 2nd passage on STS-8 is acceptable if executed properly. The CFES buffer has ionic strength that is barely compatible with cell viability and membrane stability, as seen in experiments with human erythrocytes and trypan-blue staining of human kidney cells. Cells suspended in 10% dialysed horse serum for 3 days in the cold appear to be more stable than freshly trypsinized cells. 10% horse serum appears to be superior to 5% horse serum for this purpose. The mean absolute raw mobility of HEK-8514 cells in CFES buffer at 6 degrees, conductivity 0.055 mmho/cm, is 1.1 to 1.4 um-cm/V-sec, with a range of nearly a whole mobility unit.

  18. A New Conductivity Detector for Capillary Electrophoresis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A new conductivity detector for capillary electrophoresis consisting of an electrochemical cell and a conductive meter was developed. In the cell, the microelectrode and capillary were inserted through the cell wall and fixed by screws and sealing ring, the ends of microelectrode and capillary were located by a guide with two cross holes. LOD for K+ was 1.5×10-5 mol/L.

  19. Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Y.; Yang, S.T. [Ohio State Univ., Columbus, OH (United States). Dept. of Chemical Engineering

    1998-11-20

    Acetate was produced from whey lactose in batch and fed-batch fermentations using co-immobilized cells of Clostridium formicoaceticum and Lactococcus lactis. The cells were immobilized in a spirally wound fibrous sheet packed in a 0.45-L column reactor, with liquid circulated through a 5-L stirred-tank fermentor. Industrial-grade nitrogen sources, including corn steep liquor, casein hydrolysate, and yeast hydrolysate, were studied as inexpensive nutrient supplements to whey permeate and acid whey. Supplementation with either 2.5% (v/v) corn steep liquor or 1.5 g/L casein hydrolysate was adequate for the cocultured fermentation. The overall acetic acid yield from lactose was 0.9 g/g, and the productivity was 0.25 g/(L h). Both lactate and acetate at high concentrations inhibited the homoacetic fermentation. To overcome these inhibitions, fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate. The final acetate concentration obtained in the fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate. The final acetate concentration obtained in the fed-batch fermentation was 75 g/L, which was the highest acetate concentration ever produced by C. formicoaceticum. Even at this high acetate concentration, the overall productivity was 0.18 g/(L h) based on the total medium volume and 1.23 g/(L h) based on the fibrous-bed reactor volume. The cells isolated from the fibrous-bed bioreactor at the end of this study were more tolerant to acetic acid than the original culture used to seed the bioreactor, indicating that adaptation and natural selection of acetate-tolerant strains occurred. This cocultured fermentation process could be used to produce a low-cost acetate deicer from whey permeate and acid whey.

  20. Evaluation of DNA damage in agricultural workers exposed to pesticides using single cell gel electrophoresis (comet assay

    Directory of Open Access Journals (Sweden)

    Raminderjeet Kaur

    2011-01-01

    Full Text Available Background : Pesticides are used in agriculture to protect crops, but they pose a potential risk to farmers and environment. The aim of the present study is to investigate the relation between the occupational exposure to various pesticides and the presence of DNA damage. Materials and Methods : Blood samples of 210 exposed workers (after a day of intense spraying and 50 control subjects belonging to various districts of Punjab (India were evaluated using Comet assay. Sixty workers who showed DNA damage were selected for follow up at 5-6 months after the first sampling during a low or null spraying period. Results : Significant differences were found in DNA damage between freshly exposed workers and controls and freshly exposed and followed up cases. There was significant increase in the comet parameters viz. mean comet tail length and frequency of cells showing migration in exposed workers as compared to controls (72.22 ± 20.76 vs. 46.92 ± 8.17, P<0.001; 31.79 vs. 5.77, P<0.001. In the second samples, followed up cases showed significant decrease in frequency of damaged cells as compared to freshly exposed workers of first sampling (P<0.05. The confounding factors such as variable duration of pesticide exposure, age, smoking, drinking and dietary habits etc which were expected to modulate the damage, were instead found to have no significant effect on DNA fragmentation. Conclusion : The evidence of a genetic hazard related to exposure resulting from the intensive use of pesticides stresses the need for educational programs for agricultural workers to reduce the use of chemicals in agriculture.

  1. Purification of arogenate dehydrogenase from Phenylobacterium immobile.

    Science.gov (United States)

    Mayer, E; Waldner-Sander, S; Keller, B; Keller, E; Lingens, F

    1985-01-07

    Phenylobacterium immobile, a bacterium which is able to degrade the herbicide chloridazon, utilizes for L-tyrosine synthesis arogenate as an obligatory intermediate which is converted in the final biosynthetic step by a dehydrogenase to tyrosine. This enzyme, the arogenate dehydrogenase, has been purified for the first time in a 5-step procedure to homogeneity as confirmed by electrophoresis. The Mr of the enzyme that consists of two identical subunits amounts to 69000 as established by gel electrophoresis after cross-linking the enzyme with dimethylsuberimidate. The Km values were 0.09 mM for arogenate and 0.02 mM for NAD+. The enzyme has a high specificity with respect to its substrate arogenate.

  2. Differential in Gel Electrophoresis (DIGE Comparative Proteomic Analysis of Macrophages Cell Cultures in Response to Perthamide C Treatment

    Directory of Open Access Journals (Sweden)

    Raffaele Riccio

    2013-04-01

    Full Text Available Secondary metabolites contained in marine organisms disclose diverse pharmacological activities, due to their intrinsic ability to recognize bio-macromolecules, which alter their expression and modulate their function. Thus, the identification of the cellular pathways affected by marine natural products is crucial to provide important functional information concerning their mechanism of action at the molecular level. Perthamide C, a marine sponge metabolite isolated from the polar extracts of Theonella swinhoei and endowed with a broad and interesting anti-inflammatory profile, was found in a previous study to specifically interact with heat shock protein-90 and glucose regulated protein-94, also disclosing the ability to reduce cisplatin-mediated apoptosis. In this paper, we evaluated the effect of this compound on the whole proteome of murine macrophages cells by two-dimensional DIGE proteomics. Thirty-three spots were found to be altered in expression by at least 1.6-fold and 29 proteins were identified by LC ESI-Q/TOF-MS. These proteins are involved in different processes, such as metabolism, structural stability, protein folding assistance and gene expression. Among them, perthamide C modulates the expression of several chaperones implicated in the folding of proteins correlated to apoptosis, such as Hsp90 and T-complexes, and in this context our data shed more light on the cellular effects and pathways altered by this marine cyclo-peptide.

  3. Characteristics of draft tube gas-liquid-solid fluidized-bed bioreactor with immobilized living cells for phenol degradation.

    Science.gov (United States)

    Fan, L S; Fujie, K; Long, T R; Tang, W T

    1987-09-01

    Biological phenol degradation in a draft tube gas-liquid-solid fluidized bed (DTFB) bioreactor containing a mixed culture immobilized on spherical activated carbon particles was investigated. The characteristics of biofilms including the biofilm dry density and thickness, the volumetric oxygen mass transfer coefficient, and the phenol removal rates under different operating conditions in the DTFB were evaluated. A phenol degradation rate as high as 18 kg/m(3)-day with an effluent phenol concentration less than 1 g/m(3) was achieved, signifying the high treatment efficiency of using a DTFB.

  4. Electron tomography of cryo-immobilized plant tissue: a novel approach to studying 3D macromolecular architecture of mature plant cell walls in situ.

    Science.gov (United States)

    Sarkar, Purbasha; Bosneaga, Elena; Yap, Edgar G; Das, Jyotirmoy; Tsai, Wen-Ting; Cabal, Angelo; Neuhaus, Erica; Maji, Dolonchampa; Kumar, Shailabh; Joo, Michael; Yakovlev, Sergey; Csencsits, Roseann; Yu, Zeyun; Bajaj, Chandrajit; Downing, Kenneth H; Auer, Manfred

    2014-01-01

    Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼ 2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the

  5. Electron tomography of cryo-immobilized plant tissue: a novel approach to studying 3D macromolecular architecture of mature plant cell walls in situ.

    Directory of Open Access Journals (Sweden)

    Purbasha Sarkar

    Full Text Available Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼ 2 nm, and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF, cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we

  6. Nitrogenase activity of immobilized Azotobacter vinelandii.

    Science.gov (United States)

    Seyhan, E; Kirwan, D J

    1979-02-01

    As part of a program to investigate the use of biological nitrogen fixation for fertilizer ammonia production, an investigation into the immobilization of the aerobic, nitrogen-fixing bacterium, Azotobacter vinelandii was undertaken. Immobilization was acaccomplished by adsorption onto an anionic exchange cellulose (Cellex E) with loadings as high as 10'' cells/g resin. Immobilized cell preparations were tested under both batch and continuous-flow conditions. Nitrogenase activities as high as 4200 nmol/min g resin were observed as measured by the acetylene reduction assay. Immobilized cells retained their activity for as long as 117 hr in a continuous-flow reactor. Activity loss appeared to be related to the development of a variant strain.

  7. Preparative electrophoresis for space

    Science.gov (United States)

    Rhodes, Percy H.; Snyder, Robert S.

    1988-01-01

    A premise of continuous flow electrophoresis is that removal of buoyance-induced thermal convection caused by axial and lateral temperature gradients results in ideal performance of these instruments in space. Although these gravity dependent phenomena disturb the rectilinear flow in the separation chamber when high voltage gradients or thick chamber are used, distortion of the injected sample stream due to electrodynamic effects cause major broadening of the separated bands. The electrophoresis separation process is simple, however flow local to the sample filament produced by the applied electric field were not considered. These electrohydrodynamic flows distort the sample stream and limit the separation. Also, electroosmosis and viscous flow combine to further distort the process. A moving wall concept is being proposed for space which will eliminate and control the disturbances. The moving wall entrains the fluid to move as a rigid body and produces a constant residence time for all samples distributed across the chamber thickness. The moving wall electrophoresis chamber can only be operated in space because there is no viscous flow in the chamber to stabilize against thermal convection.

  8. Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor.

    Science.gov (United States)

    Huang, Y; Yang, S T

    1998-11-20

    Acetate was produced from whey lactose in batch and fed-batch fermentations using co-immobilized cells of Clostridium formicoaceticum and Lactococcus lactis. The cells were immobilized in a spirally wound fibrous sheet packed in a 0.45-L column reactor, with liquid circulated through a 5-L stirred-tank fermentor. Industrial-grade nitrogen sources, including corn steep liquor, casein hydrolysate, and yeast hydrolysate, were studied as inexpensive nutrient supplements to whey permeate and acid whey. Supplementation with either 2.5% (v/v) corn steep liquor or 1.5 g/L casein hydrolysate was adequate for the cocultured fermentation. The overall acetic acid yield from lactose was 0.9 g/g, and the productivity was 0.25 g/(L h). Both lactate and acetate at high concentrations inhibited the homoacetic fermentation. To overcome these inhibitions, fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate. The final acetate concentration obtained in the fed-batch fermentation was 75 g/L, which was the highest acetate concentration ever produced by C. formicoaceticum. Even at this high acetate concentration, the overall productivity was 0.18 g/(L h) based on the total medium volume and 1.23 g/(L h) based on the fibrous-bed reactor volume. The cells isolated from the fibrous-bed bioreactor at the end of this study were more tolerant to acetic acid than the original culture used to seed the bioreactor, indicating that adaptation and natural selection of acetate-tolerant strains occurred. This cocultured fermentation process could be used to produce a low-cost acetate deicer from whey permeate and acid whey.

  9. Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and Saccharomyces cerevisiae Cells in Dual Matrices.

    Science.gov (United States)

    Kokkiligadda, Anusha; Beniwal, Arun; Saini, Priyanka; Vij, Shilpa

    2016-08-01

    Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.

  10. Single-cell gel electrophoresis assay in the ten spotted live-bearer fish, Cnesterodon decemmaculatus (Jenyns, 1842), as bioassay for agrochemical-induced genotoxicity.

    Science.gov (United States)

    Vera-Candioti, Josefina; Soloneski, Sonia; Larramendy, Marcelo L

    2013-12-01

    The ability of two 48 percent chlorpyrifos-based insecticides (Lorsban* 48E® and CPF Zamba®), two 50 percent pirimicarb-based insecticides (Aficida® and Patton Flow®), and two 48 percent glyphosate-based herbicides (Panzer® and Credit®) to induce DNA single-strand breaks in peripheral blood erythrocytes of Cnesterodon decemmaculatus (Jenyns, 1842) (Pisces, Poeciliidae) exposed under laboratory conditions was evaluated by the single-cell gel electrophoresis (SCGE) assay. In those fish exposed to Lorsban* 48E®, CPF Zamba®, Aficida®, Patton Flow®, Credit®, and Panzer®, a significant increase of the genetic damage was observed for all formulations regardless of the harvesting time. This genotoxic effect was achieved by an enhancement of Type II-IV comets and a concomitant decrease of Type 0-I comets over control values. A regression analysis revealed that the damage varied as a negative function of the exposure time in those Lorsban* 48E®- and Aficida®-treated fish. On the other hand, a positive correlation between damage increase and exposure time was achieved after Patton Flow® and Credit® treatment. Finally, no correlation was observed between increase in the genetic damage and exposure time after treatment with CPF Zamba® or Panzer®. These results highlight that all agrochemicals inflict primary genotoxic damage at the DNA level at sublethal concentrations, regardless of the exposure time of the aquatic organisms under study, at least within a period of 96 h of treatment.

  11. Biological hydrogen production by immobilized cells of Clostridium tyrobutyricum JM1 isolated from a food waste treatment process.

    Science.gov (United States)

    Jo, Ji Hye; Lee, Dae Sung; Park, Donghee; Park, Jong Moon

    2008-09-01

    A fermentative hydrogen-producing bacterium, Clostridium tyrobutyricum JM1, was isolated from a food waste treating process using 16S rRNA gene sequencing and amplified ribosomal DNA restriction analysis (ARDRA). A fixed-bed bioreactor packed with polyurethane foam as support matrix for the growth of the isolate was operated at different hydraulic retention time (HRT) to evaluate its performance for hydrogen production. The reactor achieved the maximal hydrogen production rate of 7.2 l H(2)l(-1)d(-1) at 2h HRT, where hydrogen content in biogas was 50.0%, and substrate conversion efficiency was 97.4%. The maximum hydrogen yield was 223 ml (g-hexose)(-1) with an influent glucose concentration of 5 g l(-1). Therefore, the immobilized reactor using C. tyrobutyricum JM1 was an effective and stable system for continuous hydrogen production.

  12. The alkaline single cell electrophoresis assay with eight mouse organs: results with 22 mono-functional alkylating agents (including 9 dialkyl N-nitrosoamines) and 10 DNA crosslinkers.

    Science.gov (United States)

    Tsuda, S; Matsusaka, N; Madarame, H; Miyamae, Y; Ishida, K; Satoh, M; Sekihashi, K; Sasaki, Y F

    2000-04-13

    The genotoxicity of 22 mono-functional alkylating agents (including 9 dialkyl N-nitrosoamines) and 10 DNA crosslinkers selected from IARC (International Agency for Research on Cancer) groups 1, 2A, and 2B was evaluated in eight mouse organs with the alkaline single cell gel electrophoresis (SCGE) (comet) assay. Groups of four mice were treated once intraperitoneally at the dose at which micronucleus tests had been conducted, and the stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were sampled 3, 8, and/or 24 h later. All chemicals were positive in the SCGE assay in at least one organ. Of the 22 mono-functional alkylating agents, over 50% were positive in all organs except the brain and bone marrow. The two subsets of mono-functional alkylating agents differed in their bone marrow genotoxicity: only 1 of the 9 dialkyl N-nitrosoamines was positive in bone marrow as opposed to 8 of the 13 other alkylating agents, reflecting the fact that dialkyl N-nitrosoamines are poor micronucleus inducers in hematopoietic cells. The two groups of mono-functional alkylating agents also differ in hepatic carcinogenicity in spite of the fact that they are similar in hepatic genotoxicity. While dialkyl N-nitrosoamines produce tumors primarily in mouse liver, only one (styrene-7,8-oxide) out of 10 of the other type of mono-functional alkylating agents is a mouse hepatic carcinogen. Taking into consideration our previous results showing high concordance between hepatic genotoxicity and carcinogenicity for aromatic amines and azo compounds, a possible explanation for the discrepancy might be that chemicals that require metabolic activation show high concordance between genotoxicity and carcinogenicity in the liver. A high percent of the 10 DNA crosslinkers were positive in the SCGE assay in the gastrointestinal mucosa, but less than 50% were positive in the liver and lung. In this study, we allowed 10 min alkali-unwinding to obtain low and stable control values

  13. Immobilization of biomacromolecules on poly-L-lactide surface via a layer-by-layer method for the improving of its cytocompatibility to bone marrow stromal cells

    Institute of Scientific and Technical Information of China (English)

    L(U) Delong; MENG Sheng; ZHONG Wei; DU Qiangguo; GONG Li; LIU Jinfen; Dusan Bakos

    2005-01-01

    Hyaluronic acid (HA) and chitosan (CS) were immobilized on the surface of poly-L-lactide (PLLA) by the following procedure: Firstly, PLLA was aminolyzed with 1, 6-hexanediamine, and part of the PLLA surface ester groups were converted to free amino groups. Then negatively charged hyaluronic acid and positively charged chitosan were deposited onto the surface of aminolyzed PLLA film in a layer-by-layer assembly manner. The effect of the layer-by- layer deposition was evaluated by ATR-FTIR spectroscopy, Raman spectroscopy and static contact angle measurements. The cytocompatibility of PLLA sample to bone marrow stromal cells (BMSCs) was improved after modification with chitosan and HA. The cell attachment, activity, and proliferation on CS/HA modified PLLA films were enhanced comparing with the control. The cells cultured on the modified PLLA samples excreted abundant cytoplasm and can differentiate to vascular smooth muscle (SM)-like (SM-like) cells. A macroporous three-dimensional PLLA scaffold was prepared by integrating both the technique of freeze-drying and particle leaching. Layer-by-layer modification by HA/CS and cell culture was also applied on this scaffold. The scaffold cultured with BMSCs for 2 weeks has been tested successfully in vivo as a patch for repairing the artificial incision on canine pulmonary artery.

  14. Immobilization Technologies in Probiotic Food Production

    OpenAIRE

    Gregoria Mitropoulou; Viktor Nedovic; Arun Goyal; Yiannis Kourkoutas

    2013-01-01

    Various supports and immobilization/encapsulation techniques have been proposed and tested for application in functional food production. In the present review, the use of probiotic microorganisms for the production of novel foods is discussed, while the benefits and criteria of using probiotic cultures are analyzed. Subsequently, immobilization/encapsulation applications in the food industry aiming at the prolongation of cell viability are described together with an evaluation of their poten...

  15. Immobilization Technologies in Probiotic Food Production

    Directory of Open Access Journals (Sweden)

    Gregoria Mitropoulou

    2013-01-01

    Full Text Available Various supports and immobilization/encapsulation techniques have been proposed and tested for application in functional food production. In the present review, the use of probiotic microorganisms for the production of novel foods is discussed, while the benefits and criteria of using probiotic cultures are analyzed. Subsequently, immobilization/encapsulation applications in the food industry aiming at the prolongation of cell viability are described together with an evaluation of their potential future impact, which is also highlighted and assessed.

  16. Characterisation of ribosomal proteins from HeLa and Krebs II mouse ascites tumor cells by different two-dimensional polyacrylamide gel electrophoresis techniques

    DEFF Research Database (Denmark)

    Issinger, O G; Beier, H

    1978-01-01

    Electrophoresis of ribosomal proteins according to Kaltschmidt and Wittmann, 1970a, b (pH 8.6/pH 4.5 urea system) yielded 29 proteins for the small subunits and 35 and 37 proteins for the large subunits of Krebs II ascites and HeLa ribosomes, respectively. Analysis of the proteins according to a ...

  17. Major proteins in normal human lymphocyte subpopulations separated by fluorescence-activated cell sorting and analyzed by two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Madsen, P S; Hokland, M; Ellegaard, J;

    1988-01-01

    We have compared the overall patterns of protein synthesis of normal human lymphocyte subpopulations taken from five volunteers using high resolution two-dimensional gel electrophoresis. The lymphocytes were isolated using density gradient centrifugation, labeled with subtype-specific MoAbs, and ...

  18. Antibody enhancement of free-flow electrophoresis

    Science.gov (United States)

    Cohly, H. H. P.; Morrison, Dennis R.; Atassi, M. Zouhair

    1988-01-01

    Specific T cell clones and antibodies (ABs) were developed to study the efficiency of purifying closely associated T cells using Continuous Flow Electrophoresis System. Enhanced separation is accomplished by tagging cells first with ABs directed against the antigenic determinants on the cell surface and then with ABs against the Fc portion of the first AB. This second AB protrudes sufficiently beyond the cell membrane and glycocalyx to become the major overall cell surface potential determinant and thus causes a reduction of electrophoretic mobility. This project was divided into three phases. Phase one included development of specific T cell clones and separation of these specific clones. Phase two extends these principles to the separation of T cells from spleen cells and immunized lymph node cells. Phase three applies this double antibody technique to the separation of T cytotoxic cells from bone marrow.

  19. Immobilizing live Escherichia coli for AFM studies of surface dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Lonergan, N.E.; Britt, L.D.; Sullivan, C.J., E-mail: sullivcj@evms.edu

    2014-02-01

    Atomic force microscopy (AFM) is a probe-based technique that permits high resolution imaging of live bacterial cells. However, stably immobilizing cells to withstand the probe-based lateral forces remains an obstacle in AFM mediated studies, especially those of live, rod shaped bacteria in nutrient media. Consequently, AFM has been under-utilized in the research of bacterial surface dynamics. The aim of the current study was to immobilize a less adherent Escherichia coli strain in a method that both facilitates AFM imaging in nutrient broth and preserves overall cell viability. Immobilization reagents and buffers were systematically evaluated and the cell membrane integrity was monitored in all sample preparations. As expected, the biocompatible gelatin coated surfaces facilitated stable cell attachment in lower ionic strength buffers, yet poorly immobilized cells in higher ionic strength buffers. In comparison, poly-L-lysine surfaces bound cells in both low and high ionic strength buffers. The benefit of the poly-L-lysine binding capacity was offset by the compromised membrane integrity exhibited by cells on poly-L-lysine surfaces. However, the addition of divalent cations and glucose to the immobilization buffer was found to mitigate this unfavorable effect. Ultimately, immobilization of E. coli cells on poly-L-lysine surfaces in a lower ionic strength buffer supplemented with Mg{sup 2+} and Ca{sup 2+} was determined to provide optimal cell attachment without compromising the overall cell viability. Cells immobilized in this method were stably imaged in media through multiple division cycles. Furthermore, permeability assays indicated that E. coli cells recover from the hypoosmotic stress caused by immobilization in low ionic strength buffers. Taken together, this data suggests that stable immobilization of viable cells on poly-L-lysine surfaces can be accomplished in lower ionic strength buffers that are supplemented with divalent cations for membrane

  20. Surface chemical immobilization of parylene C with thermosensitive block copolymer brushes based on N-isopropylacrylamide and N-tert-butylacrylamide: synthesis, characterization, and cell adhesion/detachment.

    Science.gov (United States)

    Zhang, Changhong; Vernier, P Thomas; Wu, Yu-Hsuan; Yang, Wangrong; Thompson, Mark E

    2012-01-01

    Poly(N-isopropylacrylamide) (pNIPAM), poly(N-tert-butylacrylamide) (pNTBAM), and their copolymer brushes were covalently immobilized onto parylene C (PC) surfaces via surface initiated atom transfer radical polymerization (ATRP). Contact angle measurement between 13 and 40°C showed that the hydrophobicity of the modified PC surfaces was thermally sensitive. Among these samples, PC grafted with pNIPAM (PC-NI), PC grafted with pNTBAM (PC-NT) and PC grafted with copolymer brushes containing pNTBAM and pNIPAM (PC-NT-NI) exhibited the lower critical solution temperature (LCST) at 29, 22, and 24°C, respectively. Cytocompatibility study for the modified surfaces was performed by 5 days human skin fibroblast culture at 37°C. Data showed that only a very small amount of cells adhered on the PC and PC-NI surfaces, while a significantly higher amount of cell adhesion and growth was observed on PC-NT and PC-NT-NI surfaces. Furthermore, cell detachment at the temperatures of 24 and 6°C were studied after the substrates were cultured with cells at 37°C for 24 h. The results showed that the cells on PC-NI formed the aggregations and loosely attached on the substrate after 30-min culture at 24°C, while no significant cell detachment was observed for PC-NT and PC-NT-NI samples at this temperature. By continuing the cell culture for additional 100 min at 6°C for PC-NT and PC-NT-NI, about 10 and 35% of the cells were found detached respectively, and the unattached cells aggregated on the substrate. In comparison, cells cultured on the tissue culture petri dish (TCP) exhibited no quantity and morphology changes at the culture temperatures of 37, 24, and 6°C. This study showed that: (1) immobilization of PC with nonthermal sensitive pNTBAM could provide PC surface thermal sensitive hydrophilicity; (2) the chlorines on the polymer brushes of PC-NT could be used to further initiate the ATRP pNIPAM and form block copolymer brushes; (3) the incorporation of pNTBAM into pNIPAM on PC

  1. Impact of Solvent pH on Direct Immobilization of Lysosome-Related Cell Organelle Extracts on TiO₂ for Melanin Treatment.

    Science.gov (United States)

    Bang, Seung Hyuck; Kim, Pil; Oh, Suk-Jung; Kim, Yang-Hoon; Min, Jiho

    2015-05-01

    Techniques for immobilizing effective enzymes on nanoparticles for stabilization of the activity of free enzymes have been developing as a pharmaceutical field. In this study, we examined the effect of three different pH conditions of phosphate buffer, as a dissolving solvent for lysosomal enzymes, on the direct immobilization of lysosomal enzymes extracted from Hen's egg white and Saccharomyces cerevisiae. Titanium(IV) oxide (TiO2) nanoparticles, which are extensively used in many research fields, were used in this study. The lysosomal enzymes immobilized on TiO2 under each pH condition were evaluated to maintain the specific activity of lysosomal enzymes, so that we can determine the degree of melanin treatment in lysosomal enzymes immobilized on TiO2. We found that the immobilization efficiency and melanin treatment activity in both lysosomal enzymes extracted from Hen's egg white and S. cerevisiae were the highest in an acidic condition of phosphate buffer (pH 4). However, the immobilization efficiency and melanin treatment activity were inversely proportional to the increase in pH under alkaline conditions. In addition, enhanced immobilization efficiency was shown in TiO2 pretreated with a divalent, positively charged ion, Ca(2+), and the melanin treatment activity of immobilized lysosomal enzymes on TiO2 pretreated with Ca(2+) was also increased. Therefore, this result suggests that the immobilization efficiency and melanin treatment activity of lysosomal enzymes can be enhanced according to the pH conditions of the dissolving solvent.

  2. Removal of metal cyanides from aqueous solutions by suspended and immobilized cells of rhizopus oryzae (MTCC 2541)

    Energy Technology Data Exchange (ETDEWEB)

    Roshan Dash, Rajesh; Kumar, Arvind [Department of Civil Engineering, IIT Roorkee, Roorkee, Uttarakhand (India); Balomajumder, Chandrajit [Department of Chemical Engineering, IIT Roorkee, Roorkee, Uttarakhand (India)

    2009-02-15

    This paper presents a study on biodegradation and simultaneous adsorption and biodegradation (SAB) of zinc and iron cyanides by Rhizopus oryzae (MTCC 2541), with a brief process review. Granular activated carbon was used for the immobilization of Rhizopus oryzae (MTCC 2541) for the SAB study. pH and temperature were optimized at an initial cyanide concentration of 100 mg/L for biodegradation and SAB. The microbes adapted to grow at maximum cyanide concentration were harvested and their ability to degrade cyanide was measured in both biodegradation and SAB. The removal efficiency of the SAB process was found to be better as compared to the biodegradation process. In the case of biodegradation, removal was found up to a maximum cyanide concentration of 250 mg CN{sup -}/L for zinc cyanide and 200 mg CN{sup -}/L for iron cyanide, whereas in the case of SAB, about 50% removal of cyanide at 400 mg CN{sup -}/L zinc cyanide and 300 mg CN{sup -}/L iron cyanide was possible. It was found that the SAB process is more effective than biodegradation. (Abstract Copyright [2009], Wiley Periodicals, Inc.)

  3. Purification of cell culture-derived influenza virus A/Puerto Rico/8/34 by membrane-based immobilized metal affinity chromatography.

    Science.gov (United States)

    Opitz, Lars; Hohlweg, Jonas; Reichl, Udo; Wolff, Michael W

    2009-11-01

    The presented study focuses on the feasibility of immobilized metal affinity chromatography for purification of Madin Darby canine kidney cell culture-derived influenza virus particles. Therefore, influenza virus A/Puerto Rico/8/34 was screened for adsorption to different transition metal ions attached to iminodiacetic acid. Subsequently, capturing of the same virus strain using zinc-modified iminodiacetic acid membrane adsorbers was characterized regarding viral recoveries, host cell nucleic acid and total protein depletion as well as zinc-ion-leaching. In addition, the effect of the imidazole proton pump on virus stability was studied based on the hemagglutination activity. During adsorption in the presence of 1M sodium chloride the majority of virus particles were recovered in the product (64% hemagglutination activity). Host cell nucleic acid and total protein content were reduced to approximately 7 and 26%, respectively. This inexpensive and rapid method was applied reproducibly for influenza virus A/Puerto Rico/8/34 preparations on the laboratory scale. However, preliminary results with other virus strains indicated clearly a strong strain dependency for viral adsorption.

  4. Immobilizing gold nanoparticles in mesoporous silica covered reduced graphene oxide: a hybrid material for cancer cell detection through hydrogen peroxide sensing.

    Science.gov (United States)

    Maji, Swarup Kumar; Sreejith, Sivaramapanicker; Mandal, Amal Kumar; Ma, Xing; Zhao, Yanli

    2014-08-27

    A new kind of two-dimensional (2-D) hybrid material (RGO-PMS@AuNPs), fabricated by the immobilization of ultrasmall gold nanoparticles (AuNPs, ∼3 nm) onto sandwich-like periodic mesopourous silica (PMS) coated reduced graphene oxide (RGO), was employed for both electrocatalytic application and cancer cell detection. The hybrid-based electrode sensor showed attractive electrochemical performance for sensitive and selective nonenzymatic detection of hydrogen peroxide (H2O2) in 0.1 M phosphate buffered saline, with wide linear detection range (0.5 μM to 50 mM), low detection limit (60 nM), and good sensitivity (39.2 μA mM(-1) cm(-2)), and without any interference by common interfering agents. In addition, the sensor exhibited a high capability for glucose sensing and H2O2 detection in human urine. More interestingly, the hybrid was found to be nontoxic, and the electrode sensor could sensitively detect a trace amount of H2O2 in a nanomolar level released from living tumor cells (HeLa and HepG2). Because the hybrid presents significant properties for the detection of bioactive species and certain cancerous cells by the synergistic effect from RGO, PMS, and AuNPs, it could be able to serve as a versatile platform for biosensing, bioanalysis, and biomedical applications.

  5. 3-Chloro-1,2-propanediol biodegradation by Ca-alginate immobilized Pseudomonas putida DSM 437 cells applying different processes: mass transfer effects.

    Science.gov (United States)

    Konti, Aikaterini; Mamma, Diomi; Hatzinikolaou, Dimitios G; Kekos, Dimitris

    2016-10-01

    3-Chloro-1,2-propanediol (3-CPD) biodegradation by Ca-alginate immobilized Pseudomonas putida cells was performed in batch system, continuous stirred tank reactor (CSTR), and packed-bed reactor (PBR). Batch system exhibited higher biodegradation rates and 3-CPD uptakes compared to CSTR and PBR. The two continuous systems (CSTR and PBR) when compared at 200 mg/L 3-CPD in the inlet exhibited the same removal of 3-CPD at steady state. External mass-transfer limitations are found negligible at all systems examined, since the observable modulus for external mass transfer Ω ≪ 1 and the Biot number Bi > 1. Intra-particle diffusion resistance had a significant effect on 3-CPD biodegradation in all systems studied, but to a different extent. Thiele modulus was in the range of 2.5 in batch system, but it was increased at 11 when increasing cell loading in the beads, thus lowering significantly the respective effectiveness factor. Comparing the systems at the same cell loading in the beads PBR was less affected by internal diffusional limitations compared to CSTR and batch system, and, as a result, exhibited the highest overall effectiveness factor.

  6. Derivatization in Capillary Electrophoresis.

    Science.gov (United States)

    Marina, M Luisa; Castro-Puyana, María

    2016-01-01

    Capillary electrophoresis is a well-established separation technique in analytical research laboratories worldwide. Its interesting advantages make CE an efficient and potent alternative to other chromatographic techniques. However, it is also recognized that its main drawback is the relatively poor sensitivity when using optical detection. One way to overcome this limitation is to perform a derivatization reaction which is intended to provide the analyte more suitable analytical characteristics enabling a high sensitive detection. Based on the analytical step where the CE derivatization takes place, it can be classified as precapillary (before separation), in-capillary (during separation), or postcapillary (after separation). This chapter describes the application of four different derivatization protocols (in-capillary and precapillary modes) to carry out the achiral and chiral analysis of different compounds in food and biological samples with three different detection modes (UV, LIF, and MS).

  7. Electrophoresis: the march of pennies, the march of dimes.

    Science.gov (United States)

    Righetti, Pier Giorgio

    2005-06-24

    generated immobilized pH gradients and capillary zone electrophoresis (CZE), two big players that dominated the electrokinetic horizon for all the 1990s and still in vigorous use in present days. The review terminates with a glimpse, in the third millennium, onto microchip technology and hyphenated techniques, notably direct interfacing of various electrophoretic separation methods with mass spectrometry (MS).

  8. Studies on enzyme production technology of immobilized cell from F. yellowsea YS-9412-130%黄海黄杆菌YS-9412-130固定化细胞产酶技术

    Institute of Scientific and Technical Information of China (English)

    张云波; 王跃军; 洪义国; 孙谧; 刘惠; 王春波; 刘晓萍

    2000-01-01

    3种不同的包埋材料固定化YS-9412-130菌体细胞,进行半连续发酵。结果表明,用2.5%卡拉胶固定化细胞,产酶效率最高;添加明胶对产酶不利而添加豆饼粉与玉米粉后,酶活力有显著提高;卡拉胶固定化细胞发酵液中游离菌体浓度随发酵时间变化略有上升,总体水平较低。发酵液酶活随菌体浓度的增大而呈上升趋势;固定化细胞半连续发酵效率远高于游离细胞分批发酵的效率。%The cells of F. Yellowsea YS-9412-130 immobilized with three different imbedding materials are fermented semi-continuously. The result shows that the enzyme-producing rate reaches the highest value when cells are immobilized with 2.5% carrageenan, adding soy bean cake meal and cornsteep meal can improve the enzyme activity greatly, while adding gelalin is bad to enzyme activity; the concentration of free cell in the fermented broth of carrageenan-immobilized cells arises slightly with fermentation time going, however the level in general is low; the enzyme activity of fermented broth shows a rising trend with the increase of the cells concentration; the efficiency of semi-continuous fermentation of immobilized cells is much higher than that of batch-fermentation with free cells.

  9. In-situ immobilization of quantum dots in polysaccharide-based nanogels for integration of optical pH-sensing, tumor cell imaging, and drug delivery.

    Science.gov (United States)

    Wu, Weitai; Aiello, Michael; Zhou, Ting; Berliner, Alexandra; Banerjee, Probal; Zhou, Shuiqin

    2010-04-01

    We report a class of polysaccharide-based hybrid nanogels that can integrate the functional building blocks for optical pH-sensing, cancer cell imaging, and controlled drug release into a single nanoparticle system, which can offer broad opportunities for combined diagnosis and therapy. The hybrid nanogels were prepared by in-situ immobilization of CdSe quantum dots (QDs) in the interior of the pH and temperature dual responsive hydroxypropylcellulose-poly(acrylic acid) (HPC-PAA) semi-interpenetrating polymer networks. The-OH groups of the HPC chains are designed to sequester the precursor Cd(2+) ions into the nanogels as well as stabilize the in-situ formed CdSe QDs. The pH-sensitive PAA network chains are designed to induce a pH-responsive volume phase transition of the hybrid nanogels. The developed HPC-PAA-CdSe hybrid nanogels combine a strong trap emission at 741nm for sensing physicochemical environment in a pH dependent manner and a visible excitonic emission at 592nm for mouse melanoma B16F10 cell imaging. The hybrid nanogels also provide excellent stability as a drug carrier, which cannot only provide a high drug loading capacity for a model anticancer drug temozolomide, but also offer a pH-triggered sustained-release of the drug molecules in the gel network.

  10. Hydrogen Production with High Evolution Rate and High Yield by Immobilized Cells of Hydrogen-producing Bacteria Strain B49 in a Column Reactor

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To improve the hydrogen evolution rate in continuous hydrogen production of a novel fermentative hydrogen-producing bacteria strain B49 (AF481148 in EMBL), 4 % immobilized cells by polyvinyl alcohol-boric acid method, with the addition of a small amount of calcium alginate in a column reactor obtain hydrogen yield of 2.31 mol H2/mol glucose and hydrogen evolution rate of 1435.4 ml/L culture*h respectively at medium retention time of 2.0 h with a medium containing 10g glucose/L. Moreover, as the cell density in gel beads is increased to 8%, hydrogen yield and hydrogen evolution rate for 10g glucose/L are 2.34 mol H2/mol glucose and 2912.4 ml/L culture*h respectively at medium retention time of 1.0 h, and for molasses wastewater COD of 7505.9 mg/L hydrogen production potential of 205.6 ml/g COD and hydrogen evolution rate of 2057.7 ml/L culture*h at hydraulic retention time of 0.75 h are observed. In the continuous culture pH value keeps around 3.9 by self-regulating.

  11. Low pH immobilizes and kills human leukocytes and prevents transmission of cell-associated HIV in a mouse model

    Directory of Open Access Journals (Sweden)

    Markham Richard B

    2005-09-01

    results suggest that physiologic or microbicide-induced acid immobilization and killing of infected white blood cells may be effective in preventing sexual transmission of cell-associated HIV.

  12. Modulation of Selectin-Mediated Adhesion of Flowing Lymphoma and Bone Marrow Cells by Immobilized SDF-1

    Directory of Open Access Journals (Sweden)

    Elizabeth A. Hedges

    2014-08-01

    Full Text Available The α-chemokine, stromal-derived factor-1 (SDF-1, has been linked to the homing of circulating tumor cells to bone. SDF-1 is expressed by bone microvascular cells and osteoblasts and normally functions to attract blood-borne hematopoietic stem and progenitor cells to marrow. It has been shown that treatment of cancer cells with soluble SDF-1 results in a more aggressive phenotype; however, the relevance of the administration of the soluble protein is unclear. As such, a flow device was functionalized with P-selectin and SDF-1 to mimic the bone marrow microvasculature and the initial steps of cell adhesion. The introduction of SDF-1 onto the adhesive surface was found to significantly enhance the adhesion of lymphoma cells, as well as low-density bone marrow cells (LDBMC, both in terms of the number of adherent cells and the strength of cell adhesion. Thus, SDF-1 has a synergistic effect with P-selectin on cancer cell adhesion and may be sufficient to promote preferential metastasis to bone.

  13. Pulsed field gel electrophoresis on frozen tumour tissue sections.

    OpenAIRE

    Boultwood, J; Kaklamanis, L.; Gatter, K C; Wainscoat, J S

    1992-01-01

    The application of pulsed field gel electrophoresis (PFGE) to the molecular genetic analysis of solid tumours has been restricted by the requirement for whole single cells as a DNA source. A simple technique which allows for the direct analysis of histologically characterised solid tumour material by pulsed field gel electrophoresis was developed. Single frozen tissue sections obtained from colonic carcinoma specimens were embedded without further manipulation in molten, low melting temperatu...

  14. Impedance spectra of patch clamp scenarios for single cells immobilized on a lab-on-a-chip

    DEFF Research Database (Denmark)

    Alberti, Massimo; Snakenborg, Detlef; Lopacinska, Joanna M.

    2014-01-01

    and simulated impedance spectra proved that the presented method could distinguish between a cell-attached mode and a whole-cell mode even with low-quality seals. In physiological conditions, the capacitance of HeLa cells was measured to *38 pF. The first gigaseal was recorded and maintained for 40 min. Once...... membrane. After incubating the chip for 24 h, HeLa cells adhered and grew on the chip surface but did not survive when trapped on the microapertures. The microfluidic system proved to work as a micro electrophysiological analysis system, and the IS-based method can be used for further studies on the post...

  15. Hyaluronic Acid Immobilized Polyacrylamide Nanoparticle Sensors for CD44 Receptor Targeting and pH Measurement in Cells

    DEFF Research Database (Denmark)

    Sun, Honghao; Benjaminsen, Rikke Vicki; Almdal, Kristoffer

    2012-01-01

    by radical polymerization followed by post functionalization with sensor fluorophores and physically absorbed or chemically conjugated HA. Cell uptake studies showed significant uptake of HA-coated nanosensors in HeLa cells and no uptake under the same conditions without the HA targeting ligand. The p...

  16. Diffusion of Oxygen in Alginate Gels Related to the Kinetics of Methanol Oxidation by Immobilized Hansenula polymorpha Cells

    NARCIS (Netherlands)

    Hiemstra, Harry; Dijkhuizen, Lubbert; Harder, Willem

    1983-01-01

    In the yeast Hansenula polymorpha an oxygen-requiring enzyme, alcohol oxidase, catalyzes the conversion of methanol into formaldehyde. After growth on methanol cells of the organism were harvested and entrapped in barium-alginate gels. The diffusion of oxygen towards these cells is seriously hindere

  17. INDIRECT SAFETY ASSESSMENT OF ECTODERMAL MERCURY EXPOSURE BY TRADITIONAL MEDICAL FORMULATIONS ON FRESHWATER CAT FISH CLARIAS BATRACHUS USING MICRONUCLEUS ASSAY AND ALKALINE SINGLE-CELL GEL ELECTROPHORESIS (COMET ASSAY

    Directory of Open Access Journals (Sweden)

    Anand Prem Rajan

    2012-02-01

    Full Text Available Mercury and its salts are the major constituents of Ayurvedic, Chinese and Tibetan traditional formulations. The mercury is extensively reported to accumulate in food chain and cause many neurological disorders in environment. The safety assessment of mercury on the ectodermal application on animal model is rarely reported. The void in the scientific study on external exposure of mercury and its genotoxic inside the body lead to the necessity for the present study. The freshwater cat fish Clarias batrachus was used for broad specificity genotoxic indicators micronucleus assay and alkaline single-cell gel electrophoresis (comet assays. The fish was exposed to 0.03 ppm of mercuric chloride for a period of 7, 14, 28 and 35 days ectodermally. The blood sample was assayed for the genotoxicity. The results revealed undoubted DNA damage through the micronuclei and alkaline single-cell gel electrophoresis (comet assays. Hence it is concluded the usage of traditional medicines containing the mercury may be toxic at genetic level in prolonged usage.

  18. Using a water-immiscible ionic liquid to improve asymmetric reduction of 4-(trimethylsilyl-3-butyn-2-one catalyzed by immobilized Candida parapsilosis CCTCC M203011 cells

    Directory of Open Access Journals (Sweden)

    Smith Thomas J

    2009-10-01

    Full Text Available Abstract Background Whole cells are usually employed for biocatalytic reduction reactions to ensure efficient coenzyme regeneration and to avoid problems with enzyme purification and stability. The efficiency of whole cell-catalyzed bioreduction is frequently restricted by pronounced toxicity of substrate and/or product to the microbial cells and in many instances the use of two-phase reaction systems can solve such problems. Therefore, we developed new, biphasic reaction systems with biocompatible water-immiscible ionic liquids (ILs as alternatives to conventional organic solvents, in order to improve the asymmetric reduction of 4-(trimethylsilyl-3-butyn-2-one (TMSB to (S-4-(trimethylsilyl-3-butyn-2-ol {(S-TMSBOL}, a key intermediate for synthesis of 5-lipoxygenase inhibitors, using immobilized Candida parapsilosis CCTCC M203011 cells as the biocatalyst. Results Various ILs exerted significant but different effects on the bioreduction. Of all the tested water-immiscible ILs, the best results were observed with 1-butyl-3-methylimidazolium hexafluorophosphate (C4MIM·PF6, which exhibited not only good biocompatibility with the cells but also excellent solvent properties for the toxic substrate and product, thus markedly improving the efficiency of the bioreduction and the operational stability of the cells as compared to the IL-free aqueous system. 2-Propanol was shown to be the most suitable co-substrate for coenzyme regeneration, and it was found that the optimum volume ratio of buffer to C4MIM·PF6, substrate concentration, buffer pH, 2-propanol concentration and reaction temperature were 4/1 (v/v, 24 mM, 5.5, 130 mM and 30°C, respectively. Under these optimized conditions, the maximum yield and the product e.e. wer 97.7% and >99%, respectively, which are much higher than the corresponding values previously reported. The efficient whole-cell biocatalytic process was shown to be feasible on a 250-mL scale. Conclusion The whole cell

  19. Coupled reactions of immobilized enzymes and immobilized substrates: clinical application as exemplified by amylase assay.

    Science.gov (United States)

    Barabino, R C; Gray, D N; Keyes, M H

    1978-08-01

    We described a partitioned enzyme-sensor system, which incorporates an immoblized substrate and three or more discrete immobilized enzymes. This instrument measures alpha-amylase activity by passing the solution containing alpha-amylase over a column packed with immobilized starch. The resulting oligosaccharides are successively exposed to a column or columns containing immobolized glucose oxidase, catalase, glucoamylase or maltase, and glucose oxidase. The resulting hydrogen peroxide is detected by a three-electrode amperometric cell. All immobilized reagents were immobilized on a particulate, porous alumina to allow rapid and constant flow rate. With use of less than optimum immobilized reagents, alpha-amylase activity has been measured from about 5 to 200 kU/liter with a 50 microliter sample size. Lack of sensitivity is predominantly attributable to the low activity and low stability of immobilized maltase and glucoamylase. We believe that a clinical test using this system is feasible and desirable because the immobilized reagent system should allow for testing of alpha-amylase with excellent precision, convenience to the operator, and low cost.

  20. Predictive markers for the response to 5-fluorouracil therapy in cancer cells: Constant-field gel electrophoresis as a tool for prediction of response to 5-fluorouracil-based chemotherapy.

    Science.gov (United States)

    Saleh, E M; El-Awady, R A; Anis, N

    2013-01-01

    The prediction of response or severe toxicity and therapy individualisation are extremely important in cancer chemotherapy. There are few tools to predict chemoresponse or toxicity in cancer patients. We investigated the correlation between the induction and repair of DNA double-strand breaks (DSBs) using constant-field gel electrophoresis (CFGE) and evaluating cell cycle progression and the sensitivity of four cancer cell lines to 5-fluorouracil (5FU). Using a sulphorhodamine-B assay, colon carcinoma cells (HCT116) were found to be the most sensitive to 5FU, followed by liver carcinoma cells (HepG2) and breast carcinoma cells (MCF-7). Cervical carcinoma cells (HeLa) were the most resistant. As measured by CFGE, DSB induction, but not residual DSBs, exhibited a significant correlation with the sensitivity of the cell lines to 5FU. Flow cytometric cell cycle analysis revealed that 14% of HCT116 or HepG2 cells and 2% of MCF-7 cells shifted to sub-G1 phase after a 96-h incubation with 5FU. Another 5FU-induced cell cycle change in HCT116, HepG2 and MCF-7 cells was the mild arrest of cells in G1 and/or G2/M phases of the cell cycle. In addition, 5FU treatment resulted in the accumulation of HeLa cells in the S and G2/M phases. Determination of Fas ligand (Fas-L) and caspase 9 as representative markers for the extrinsic and intrinsic pathways of apoptosis, respectively, revealed that 5FU-induced apoptosis in HCT116 and HepG2 results from the expression of Fas-L (extrinsic pathway). Therefore, the induction of DNA DSBs by 5FU, detected using CFGE, and the induction of apoptosis are candidate predictive markers that may distinguish cancer cells which are likely to benefit from 5FU treatment and the measurement of DSBs using CFGE may aid the prediction of clinical outcome.

  1. Copolymers For Capillary Gel Electrophoresis

    Science.gov (United States)

    Liu, Changsheng; Li, Qingbo

    2005-08-09

    This invention relates to an electrophoresis separation medium having a gel matrix of at least one random, linear copolymer comprising a primary comonomer and at least one secondary comonomer, wherein the comonomers are randomly distributed along the copolymer chain. The primary comonomer is an acrylamide or an acrylamide derivative that provides the primary physical, chemical, and sieving properties of the gel matrix. The at least one secondary comonomer imparts an inherent physical, chemical, or sieving property to the copolymer chain. The primary and secondary comonomers are present in a ratio sufficient to induce desired properties that optimize electrophoresis performance. The invention also relates to a method of separating a mixture of biological molecules using this gel matrix, a method of preparing the novel electrophoresis separation medium, and a capillary tube filled with the electrophoresis separation medium.

  2. Biomedical applications of capillary electrophoresis

    Science.gov (United States)

    Kartsova, L. A.; Bessonova, E. A.

    2015-08-01

    The review deals with modern analytical approaches used in capillary electrophoresis for solving medical and biological problems: search for biomarkers of various diseases and rapid diagnosis based on characteristic profiles of biologically active compounds by capillary electrophoresis with mass spectrometric detection; monitoring of the residual drugs in biological fluids for evaluating the efficiency of drug therapy; testing of the enantiomeric purity of pharmaceutical products; the use of novel materials as components of stationary and pseudo-stationary phases in capillary electrophoresis and capillary electrochromatography to increase the selectivity of separation of components of complex matrices; and identification of various on-line preconcentration techniques to reduce the detection limits of biologically active analytes. A topical trend in capillary electrophoresis required in clinical practice, viz., the design of microfluidic systems, is discussed. The bibliography includes 173 references.

  3. DNA typing by capillary electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, N.

    1997-10-08

    Capillary electrophoresis is becoming more and more important in nucleic acid analysis including DNA sequencing, typing and disease gene measurements. This work summarized the background of DNA typing. The recent development of capillary electrophoresis was also discussed. The second part of the thesis showed the principle of DNA typing based on using the allelic ladder as the absolute standard ladder in capillary electrophoresis system. Future work will be focused on demonstrating DNA typing on multiplex loci and examples of disease diagnosis in the on-line format of PCR-CE. Also capillary array electrophoresis system should allow high throughput, fast speed DNA typing. Only the introduction and conclusions for this report are available here. A reprint was removed for separate processing.

  4. Applications of space-electrophoresis in medicine. [for cellular separations in molecular biology

    Science.gov (United States)

    Bier, M.

    1976-01-01

    The nature of electrophoresis is reviewed and potential advances realizable in the field of biology and medicine from a space electrophoresis facility are examined. The ground-based applications of electrophoresis: (1) characterization of an ionized species; (2) determination of the quantitative composition of a complex mixture; and (3) isolation of the components of a mixture, separation achieved on the basis of the difference in transport rates is reviewed. The electrophoresis of living cells is considered, touching upon the following areas: the separation of T and B lymphocytes; the genetic influence on mouse lymphocyte mobilities; the abnormal production of specific and monoclonal immunoproteins; and the study of cancer. Schematic diagrams are presented of three types of electrophoresis apparatus: the column assembly for the static electrophoresis experiment on the Apollo-Soyuz mission, the continuous flow apparatus used in the same mission and a miniaturized electrophoresis apparatus.

  5. A highly efficient immobilized ZnO/Zn photoanode for degradation of azo dye Reactive Green 19 in a photocatalytic fuel cell.

    Science.gov (United States)

    Lee, Sin-Li; Ho, Li-Ngee; Ong, Soon-An; Wong, Yee-Shian; Voon, Chun-Hong; Khalik, Wan Fadhilah; Yusoff, Nik Athirah; Nordin, Noradiba

    2017-01-01

    Photocatalytic fuel cell (PFC) is a potential wastewater treatment technology that can generate electricity from the conversion of chemical energy of organic pollutants. An immobilized ZnO/Zn fabricated by sonication and heat attachment method was applied as the photoanode and Pt/C plate was used as the cathode of the PFC in this study. Factors that affect the decolorization efficiency and electricity generation of the PFC such as different initial dye concentrations and pH were investigated. Results revealed that the degradation of Reactive Green 19 (RG19) was enhanced in a closed circuit PFC compared with that of a opened circuit PFC. Almost 100% decolorization could be achieved in 8 h when 250 mL of 30 mg L(-1) of RG19 was treated in a PFC without any supporting electrolyte. The highest short circuit current of 0.0427 mA cm(-2) and maximum power density of 0.0102 mW cm(-2) was obtained by PFC using 30 mg L(-1) of RG19. The correlation between dye degradation, conductivity and voltage output were also investigated and discussed.

  6. Immobilization of aluminum with mucilage secreted by root cap and root border cells is related to aluminum resistance in Glycine max L.

    Science.gov (United States)

    Cai, Miaozhen; Wang, Ning; Xing, Chenghua; Wang, Fangmei; Wu, Kun; Du, Xing

    2013-12-01

    The root cap and root border cells (RBCs) of most plant species produced pectinaceous mucilage, which can bind metal cations. In order to evaluate the potential role of root mucilage on aluminum (Al) resistance, two soybean cultivars differing in Al resistance were aeroponic cultured, the effects of Al on root mucilage secretion, root growth, contents of mucilage-bound Al and root tip Al, and the capability of mucilage to bind Al were investigated. Increasing Al concentration and exposure time significantly enhanced mucilage excretion from both root caps and RBCs, decreased RBCs viability and relative root elongation except roots exposed to 400 μM Al for 48 h in Al-resistant cultivar. Removal of root mucilage from root tips resulted in a more severe inhibition of root elongation. Of the total Al accumulated in root, mucilage accounted 48-72 and 12-27 %, while root tip accounted 22-52 and 73-88 % in Al-resistant and Al-sensitive cultivars, respectively. A (27)Al nuclear magnetic resonance spectrum of the Al-adsorbed mucilage showed Al tightly bound to mucilage. Higher capacity to exclude Al in Al-resistant soybean cultivar is related to the immobilization and detoxification of Al by the mucilage secreted from root cap and RBCs.

  7. Integration of continuous production and recovery of solvents from whey permeate: Use of immobilized cells of Clostridium acetobutylicum in a fluidized bed reactor coupled with gas stripping

    Energy Technology Data Exchange (ETDEWEB)

    Qureshi, N.; Maddox, I.S. (Massey Univ., Palmerston North (New Zealand). Biotechnology Dept.)

    1991-01-01

    An investigation was performed into the operation of an integrated system for continuous production and product recovery of solvents (acetone-butanol-ethanol) from the ABE fermentation process. Cells of Clostridium acetobutylicum were immobilized by adsorption onto bonechar, and used in a fluidized bed reactor for continuous solvent production from whey permeate. The reactor effluent was stripped of the solvents using nitrogen gas, and was recycled to the reactor. This relieved product inhibition and allowed further sugar utilization. At a dilution rate of 1.37 h{sup -1} a reactor productivity of 5.1 kg/(cm{sup 3}xh) was achieved. The solvents in the stripping gas were condensed to give a solution of 53.7 kg/m{sup 3}. This system has the advantages of relieving product inhibition, and providing a more concentrated solution for recovery by distillation. Residual sugar and non-volatile reaction intermediate are not removed by gas stripping and this contributes to high solvent yields. (orig.).

  8. Back propagation neural network model for predicting the performance of immobilized cell biofilters handling gas-phase hydrogen sulphide and ammonia.

    Science.gov (United States)

    Rene, Eldon R; López, M Estefanía; Kim, Jung Hoon; Park, Hung Suck

    2013-01-01

    Lab scale studies were conducted to evaluate the performance of two simultaneously operated immobilized cell biofilters (ICBs) for removing hydrogen sulphide (H2S) and ammonia (NH3) from gas phase. The removal efficiencies (REs) of the biofilter treating H2S varied from 50 to 100% at inlet loading rates (ILRs) varying up to 13 g H2S/m(3) ·h, while the NH3 biofilter showed REs ranging from 60 to 100% at ILRs varying between 0.5 and 5.5 g NH3/m(3) ·h. An application of the back propagation neural network (BPNN) to predict the performance parameter, namely, RE (%) using this experimental data is presented in this paper. The input parameters to the network were unit flow (per min) and inlet concentrations (ppmv), respectively. The accuracy of BPNN-based model predictions were evaluated by providing the trained network topology with a test dataset and also by calculating the regression coefficient (R (2)) values. The results from this predictive modeling work showed that BPNNs were able to predict the RE of both the ICBs efficiently.

  9. Back Propagation Neural Network Model for Predicting the Performance of Immobilized Cell Biofilters Handling Gas-Phase Hydrogen Sulphide and Ammonia

    Directory of Open Access Journals (Sweden)

    Eldon R. Rene

    2013-01-01

    Full Text Available Lab scale studies were conducted to evaluate the performance of two simultaneously operated immobilized cell biofilters (ICBs for removing hydrogen sulphide (H2S and ammonia (NH3 from gas phase. The removal efficiencies (REs of the biofilter treating H2S varied from 50 to 100% at inlet loading rates (ILRs varying up to 13 g H2S/m3·h, while the NH3 biofilter showed REs ranging from 60 to 100% at ILRs varying between 0.5 and 5.5 g NH3/m3·h. An application of the back propagation neural network (BPNN to predict the performance parameter, namely, RE (% using this experimental data is presented in this paper. The input parameters to the network were unit flow (per min and inlet concentrations (ppmv, respectively. The accuracy of BPNN-based model predictions were evaluated by providing the trained network topology with a test dataset and also by calculating the regression coefficient (R2 values. The results from this predictive modeling work showed that BPNNs were able to predict the RE of both the ICBs efficiently.

  10. Dynamics of yeast immobilized-cell fluidized-bed bioreactors systems in ethanol fermentation from lactose-hydrolyzed whey and whey permeate.

    Science.gov (United States)

    Gabardo, Sabrina; Pereira, Gabriela Feix; Klein, Manuela P; Rech, Rosane; Hertz, Plinho F; Ayub, Marco Antônio Záchia

    2016-01-01

    We studied the dynamics of ethanol production on lactose-hydrolyzed whey (LHW) and lactose-hydrolyzed whey permeate (LHWP) in batch fluidized-bed bioreactors using single and co-cultures of immobilized cells of industrial strains of Saccharomyces cerevisiae and non-industrial strains of Kluyveromyces marxianus. Although the co-culture of S. cerevisiae CAT-1 and K. marxianus CCT 4086 produced two- to fourfold the ethanol productivity of single cultures of S. cerevisiae, the single cultures of the K. marxianus CCT 4086 produced the best results in both media (Y EtOH/S = 0.47-0.49 g g(-1) and Q P = 1.39-1.68 g L(-1) h(-1), in LHW and LHWP, respectively). Ethanol production on concentrated LHWP (180 g L(-1)) reached 79.1 g L(-1), with yields of 0.46 g g(-1) for K. marxianus CCT 4086 cultures. Repeated batches of fluidized-bed bioreactor on concentrated LHWP led to increased ethanol productivity, reaching 2.8 g L(-1) h(-1).

  11. Asymmetric Synthesis of (—)—1—Trimethylsiyl—ethanol with Immobilized Saccharomyces Cerevisiae Cells in Water/Organic Solvent Biphasic System

    Institute of Scientific and Technical Information of China (English)

    娄文勇; 宗敏华; 范晓丹

    2003-01-01

    Asymmetric synthesis of (-)-1-trimethylsilyl-ethanol with immobilized Saccharomyces cerevisiae cells in water/organic solvent biphasic system was studied,The effects of shake speed,hydrophobictiy of organic solvent ,volume ratio of water phase to organic phase,pH value of aqueous phase and reaction temperature on the initial reaction rate,maximum yield and enantiomeric excess(ee) of the product were systematically explored,All the above-mentioned factors had significant infuence on the reaction.n-Hexane was found to be the best organic solvent for the reaction.The optimum shake speed,volume ratio of water phase to organic phase,pH value and reaction temperature were 150 r.min-1,1/2,8 and 30℃ respectively,under which the maximum yield and enantiomeric excess of the product were as high as 96.8% and 95.7%,which are 15% and 16% higher than those of the corresponding reaction performed in aqueous phase ,To our best knowledge,this is the most satisfactory result obtained.

  12. Cadmium-free sugar-chain-immobilized fluorescent nanoparticles containing low-toxicity ZnS-AgInS2 cores for probing lectin and cells.

    Science.gov (United States)

    Shinchi, Hiroyuki; Wakao, Masahiro; Nagata, Nonoka; Sakamoto, Masaya; Mochizuki, Eiko; Uematsu, Taro; Kuwabata, Susumu; Suda, Yasuo

    2014-02-19

    Sugar chains play a significant role in various biological processes through sugar chain-protein and sugar chain-sugar chain interactions. To date, various tools for analyzing sugar chains biofunctions have been developed. Fluorescent nanoparticles (FNPs) functionalized with carbohydrate, such as quantum dots (QDs), are an attractive imaging tool for analyzing carbohydrate biofunctions in vitro and in vivo. Most FNPs, however, consist of highly toxic elements such as cadmium, tellurium, selenium, and so on, causing problems in long-term bioimaging because of their cytotoxicity. In this study, we developed cadmium-free sugar-chain-immobilized fluorescent nanoparticles (SFNPs) using ZnS-AgInS2 (ZAIS) solid solution nanoparticles (NPs) of low or negligible toxicity as core components, and investigated their bioavailability and cytotoxicity. SFNPs were prepared by mixing our originally developed sugar-chain-ligand conjugates with ZAIS/ZnS core/shell NPs. In binding experiments with lectin, the obtained ZAIS/ZnS SFNPs interacted with an appropriate lectin to give specific aggregates, and their binding interaction was visually and/or spectroscopically detected. In addition, these SFNPs were successfully utilized for cytometry analysis and cellular imaging in which the cell was found to possess different sugar-binding properties. The results of the cytotoxicity assay indicated that SFNPs containing ZAIS/ZnS have much lower toxicity than those containing cadmium. These data strongly suggest that our designed SFNPs can be widely utilized in various biosensing applications involved in carbohydrates.

  13. Shear stress effects on production of exopolymeric substances and biofilm characteristics during phenol biodegradation by immobilized Pseudomonas desmolyticum (NCIM2112) cells in a pulsed plate bioreactor.

    Science.gov (United States)

    Veena, B R; Shetty K, Vidya; Saidutta, M B

    2016-07-03

    This article reports studies on a continuous pulsed plate bioreactor (PPBR) with the cells of Pseudomonas desmolyticum (NCIM2112) immobilized on granular activated carbon (GAC) used as a biofilm reactor for biodegradation of phenol. Almost complete removal of 200 ppm phenol could be achieved in this bioreactor. Biofilm structure and characteristics are influenced by hydrodynamic and shear conditions in bioreactors. In this article, the effect of shear stress induced by frequency of pulsation on biofilm characteristics during the startup period in the PPBR is reported. The startup time decreased with the increase in frequency of pulsation. The formation of biofilm in PPBR was found to have three phases: accumulation, compaction, and plateau. The effect of frequency on production of exoploymeric substances (EPS) such as, protein, carbohydrate, and humic substance is reported. An increase in shear stress induced by the frequency of pulsation increased the production of exopolymeric substances in the biofilm during startup of the bioreactor. Increase in shear stress caused a decrease in biofilm thickness and an increase in dry density of the biofilm. Increase in shear stress resulted in a smoother and thinner biofilm surface with more compact and dense structure.

  14. Immobilizing live Escherichia coli for AFM studies of surface dynamics.

    Science.gov (United States)

    Lonergan, N E; Britt, L D; Sullivan, C J

    2014-02-01

    Atomic force microscopy (AFM) is a probe-based technique that permits high resolution imaging of live bacterial cells. However, stably immobilizing cells to withstand the probe-based lateral forces remains an obstacle in AFM mediated studies, especially those of live, rod shaped bacteria in nutrient media. Consequently, AFM has been under-utilized in the research of bacterial surface dynamics. The aim of the current study was to immobilize a less adherent Escherichia coli strain in a method that both facilitates AFM imaging in nutrient broth and preserves overall cell viability. Immobilization reagents and buffers were systematically evaluated and the cell membrane integrity was monitored in all sample preparations. As expected, the biocompatible gelatin coated surfaces facilitated stable cell attachment in lower ionic strength buffers, yet poorly immobilized cells in higher ionic strength buffers. In comparison, poly-l-lysine surfaces bound cells in both low and high ionic strength buffers. The benefit of the poly-l-lysine binding capacity was offset by the compromised membrane integrity exhibited by cells on poly-l-lysine surfaces. However, the addition of divalent cations and glucose to the immobilization buffer was found to mitigate this unfavorable effect. Ultimately, immobilization of E. coli cells on poly-l-lysine surfaces in a lower ionic strength buffer supplemented with Mg(2+) and Ca(2+) was determined to provide optimal cell attachment without compromising the overall cell viability. Cells immobilized in this method were stably imaged in media through multiple division cycles. Furthermore, permeability assays indicated that E. coli cells recover from the hypoosmotic stress caused by immobilization in low ionic strength buffers. Taken together, this data suggests that stable immobilization of viable cells on poly-l-lysine surfaces can be accomplished in lower ionic strength buffers that are supplemented with divalent cations for membrane stabilization

  15. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation

    Energy Technology Data Exchange (ETDEWEB)

    Godoy-Gallardo, Maria, E-mail: maria.godoy.gallardo@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Guillem-Marti, Jordi, E-mail: jordi.guillem.marti@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Sevilla, Pablo, E-mail: psevilla@euss.es [Department of Mechanics, Escola Universitària Salesiana de Sarrià (EUSS), C/ Passeig de Sant Bosco, 42, 08017 Barcelona (Spain); Manero, José M., E-mail: jose.maria.manero@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Gil, Francisco J., E-mail: francesc.xavier.gil@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); and others

    2016-02-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria–cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties. - Highlights: • TESPSA silane induces osteoblast differentiation. • TESPSA reduces bacterial adhesion and biofilm formation. • TESPSA is a promising anchoring platform of biomolecules onto titanium.

  16. Immobilization of foreign protein into polyhedra of Bombyx mori nucleopolyhedrovirus (BmNPV)

    Institute of Scientific and Technical Information of China (English)

    Xing-wei XIANG; Rui YANG; Lin CHEN; Xiao-long HU; Shao-fang YU; Cui-ping CAO; Xiao-feng WU

    2012-01-01

    In the late phase of Bombyx mori nucleopolyhedrovirus (BmNPV) infection,a large amount of polyhedra appear in the infected cell nucleolus,these polyhedra being dense protein crystals protecting the incorporated virions from the harsh environment.To investigate whether the foreign protein could be immobilized into the polyhedra of BmNPV,two recombinant baculoviruses were generated by a novel BmNPV polyhedrin-plus (polh+) Bac-toBac system,designated as vBmBac(polh+)-enhanced green fluorescent protein (EGFP) and vBmBac(polh+)-LacZ,which can express the polyhedrin and foreign protein simultaneously.Light microscopy analysis showed that all viruses produced polyhedra of normal appearance.Green fluorescence can be apparently detected on the surface of the vBmBac(polh+)-EGFP polyhedra,but not the BmNPV polyhedra.Fluorescence analysis and anti-desiccation testing confirmed that EGFP was embedded in the polyhedra.As expected,the vBmBac(polh+)-LacZ polyhedre contained an amount of LacZ and had a higher β-galactosidase activity.Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were also performed to verify if the foreign proteins were immobilized into polyhedra.This study provides a new inspiration for efficient preservation of useful proteins and development of new pesticides with toxic proteins.

  17. Thermally-dried free and immobilized kefir cells as starter culture in hard-type cheese production.

    Science.gov (United States)

    Katechaki, Eleftheria; Panas, Panayiotis; Kourkoutas, Yiannis; Koliopoulos, Dionisis; Koutinas, Athanasios A

    2009-07-01

    In an attempt to seek for suitable dried cultures, thermally-dried kefir was employed as starter in hard-type cheese production and tested in cheeses ripened at 5, 18 and 22 degrees C. Both free and immobilised on casein kefir cells were used and compared to cheese made without starter culture. Cheese products made with free cells of kefir culture were characterized by longer preservation time, improved aroma, taste, texture characteristics and increased degree of openness. Volatile profiles obtained by GC/MS analysis revealed a 216% increase in total concentration of esters, organic acids, alcohols and carbonyl compounds between cheeses prepared with and without kefir culture.

  18. Free zone electrophoresis simulation of static column electrophoresis in microgravity on shuttle flight STS-3

    Science.gov (United States)

    Todd, P. W.; Hjerten, S.

    1985-01-01

    Experiments were designed to replicate, as closely as possible in 1-G, the conditions of the STS-3 red blood cell (RBC) experiments. Free zone electrophoresis was the method of choice, since it minimizes the role of gravity in cell migration. The physical conditions of the STS-3 experiments were used, and human and rabbit RBC's fixed by the same method were the test particles. The effects of cell concentration, electroosmotic mobility, and sample composition were tested in order to seek explanations for the STS-3 results and to provide data on cell concentration effects for future zero-G separation on the continuous-flow zero-G electrophoretics separator.

  19. THERMAL ACTIVATION OF IMMOBILIZED PAPAIN

    Institute of Scientific and Technical Information of China (English)

    1998-01-01

    Papain (Papainase, EC 3.4.22.2) was immobilized on porous silica beads by cross linking with glutaraldehyde. The thermal activation of this immobilized papain in aqueous system was found at a temperature range from 50 to 90℃. The higher the temperature, the more active the immobilized papain will possess. At the same time,the durability of the immobilized papain on heating was greatly improved. The effect of additives and salts on the activity of the immobilized papain were also studied. The results showed that the additives and some of the salts studied could markedly enhance the activity of the immobilized papain at elevated temperature.

  20. Escherichia coli NemA is an efficient chromate reductase that can be biologically immobilized to provide a cell free system for remediation of hexavalent chromium.

    Directory of Open Access Journals (Sweden)

    Katherine J Robins

    Full Text Available Hexavalent chromium is a serious and widespread environmental pollutant. Although many bacteria have been identified that can transform highly water-soluble and toxic Cr(VI to insoluble and relatively non-toxic Cr(III, bacterial bioremediation of Cr(VI pollution is limited by a number of issues, in particular chromium toxicity to the remediating cells. To address this we sought to develop an immobilized enzymatic system for Cr(VI remediation. To identify novel Cr(VI reductase enzymes we first screened cell extracts from an Escherichia coli library of soluble oxidoreductases derived from a range of bacteria, but found that a number of these enzymes can reduce Cr(VI indirectly, via redox intermediates present in the crude extracts. Instead, activity assays for 15 candidate enzymes purified as His6-tagged proteins identified E. coli NemA as a highly efficient Cr(VI reductase (k(cat/K(M= 1.1×10(5 M(-1 s(-1 with NADH as cofactor. Fusion of nemA to the polyhydroxyalkanoate synthase gene phaC from Ralstonia eutropha enabled high-level biosynthesis of functionalized polyhydroxyalkanoate granules displaying stable and active NemA on their surface. When these granules were combined with either Bacillus subtilis glucose dehydrogenase or Candida boidinii formate dehydrogenase as a cofactor regenerating partner, high levels of chromate transformation were observed with only low initial concentrations of expensive NADH cofactor being required, the overall reaction being powered by consumption of the cheap sacrificial substrates glucose or formic acid, respectively. This system therefore offers promise as an economic solution for ex situ Cr(VI remediation.

  1. Comparison of protein patterns of xrs-5, a radiosensitive Chinese hamster ovary cell line, and CHO-K1, its radioresistant parent, using two-dimensional gel-electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Kramer, J.M. (Miami Univ., Oxford, OH (USA). Dept. of Zoology)

    1991-01-01

    X-ray sensitive strains of Chinese hamster ovary cell lines have been used to analyze radiation repair mechanisms. One cell line, xrs-5, has been shown to be very sensitive to ionizing radiation and radical forming chemical mutagens. This sensitivity is thought to be a result a mutation in the DNA double strand break (DSB) repair mechanism, and its characterization has been a goal of several repair mechanism studies. Using two-dimensional gel electrophoresis, we have detected a protein (MW approximately 55KD) in the DNA/Nuclear Matrix (nucleoid) cell fraction of CHO-Kl cells that is absent in the nucleoid fraction of xrs-5. This protein is present, however, in both CHO-Kl and xrs-5 whole cell protein maps. To determine whether the 55KD protein is responsible for the radiosensitive and defective DSB repair phenotype of xrs-5 cells, studies are now underway to analyze revertants of xrs-5 that are proficient in DSB repair. Furthermore, an effort to sequence the protein in question is planned. 23 refs., 2 figs.

  2. Green biosynthesis of floxuridine by immobilized microorganisms.

    Science.gov (United States)

    Rivero, Cintia W; Britos, Claudia N; Lozano, Mario E; Sinisterra, Jose V; Trelles, Jorge A

    2012-06-01

    This work describes an efficient, simple, and green bioprocess for obtaining 5-halogenated pyrimidine nucleosides from thymidine by transglycosylation using whole cells. Biosynthesis of 5-fluoro-2'-deoxyuridine (floxuridine) was achieved by free and immobilized Aeromonas salmonicida ATCC 27013 with an 80% and 65% conversion occurring in 1 h, respectively. The immobilized biocatalyst was stable for more than 4 months in storage conditions (4 °C) and could be reused at least 30 times without loss of its activity. This microorganism was able to biosynthesize 2.0 mg L(-1) min(-1) (60%) of 5-chloro-2'-deoxyuridine in 3 h. These halogenated pyrimidine 2'-deoxynucleosides are used as antitumoral agents.

  3. Biomolecule immobilization techniques for bioactive paper fabrication.

    Science.gov (United States)

    Kong, Fanzhi; Hu, Yim Fun

    2012-04-01

    Research into paper-based sensors or functional materials that can perform analytical functions with active recognition capabilities is rapidly expanding, and significant research effort has been made into the design and fabrication of bioactive paper at the biosensor level to detect potential health hazards. A key step in the fabrication of bioactive paper is the design of the experimental and operational procedures for the immobilization of biomolecules such as antibodies, enzymes, phages, cells, proteins, synthetic polymers and DNA aptamers on a suitably prepared paper membrane. The immobilization methods are concisely categorized into physical absorption, bioactive ink entrapment, bioaffinity attachment and covalent chemical bonding immobilization. Each method has individual immobilization characteristics. Although every biomolecule-paper combination has to be optimized before use, the bioactive ink entrapment method is the most commonly used approach owing to its general applicability and biocompatibility. Currently, there are four common applications of bioactive paper: (1) paper-based bioassay or paper-based analytical devices for sample conditioning; (2) counterfeiting and countertempering in the packaging and construction industries; (3) pathogen detection for food and water quality monitoring; and (4) deactivation of pathogenic bacteria using antimicrobial paper. This article reviews and compares the different biomolecule immobilization techniques and discusses current trends. Current, emerging and future applications of bioactive paper are also discussed.

  4. Effect of cell immobilization on the growth dynamics of Salmonella Typhimurium and Escherichia coli at suboptimal temperatures.

    Science.gov (United States)

    Smet, Cindy; Van Derlinden, Eva; Mertens, Laurence; Noriega, Estefanía; Van Impe, Jan F

    2015-09-02

    Predictive microbiology has recently acknowledged the impact of the solid(like) food structure on microbial behavior. The presence of this solid(like) structure causes microorganisms to grow as colonies and no longer planktonically as in liquid. In this paper, the growth dynamics of Salmonella Typhimurium and Escherichia coli were studied as a function of temperature, considering different growth morphologies, i.e., (i) planktonic cells, (ii) immersed colonies and (iii) surface colonies. For all three growth morphologies, both microorganisms were grown in petri dishes. While E. coli was grown under optimal pH and water activity (aw), for S. Typhimurium pH and aw were adapted to 5.5 and 0.990. In order to mimic a solid(like) environment, 5% (w/v) gelatin was added. All petri dishes were incubated under static conditions at temperatures in the range [8.0°C-22.0°C]. Cell density was determined via viable plate counting. This work demonstrates that the growth morphology (planktonic vs. colony) has a negligible effect on the growth dynamics as a function of temperature. The observation of almost equal growth rates for planktonic cultures and colonies is in contrast to literature where, mostly, a difference is observed, i.e., μplanktonic cells≥μimmersed colonies≥μsurface colonies. This difference might be due to shaking of the liquid culture in these studies, which results in a nutrient and oxygen rich environment, in contrast to the diffusion-limited gel system. Experiments also indicate that lag phases for solid(like) systems are similar to those for the planktonic cultures, as can be found in literature for similar growth conditions. Considering the maximum cell density, no clear trend was deducted for either of the microorganisms. This study indicates that the growth parameters in the suboptimal temperature range do not depend on the growth morphology. For the considered experimental conditions, models previously developed for liquid environments can be used

  5. Electrophoresis as a management tool

    Science.gov (United States)

    Morgan, R.P.; Chapman, J.A.; Noe, L.A.; Henny, C.J.

    1974-01-01

    The theme of this 1974 Northeast Fish and Wildlife Conference is 'A New Era'. Indeed, it is a new era for improved techniques to assist in management of our fish and wildlife resources for the maximum benefit of all. In some cases, the new techniques are primarily used in research.on fish and wildlife, and the results from the research are used to aid management and enforcement agencies in the decision-making process. One of the newer techniques that is being applied to problems in fisheries and wildlife is electrophoresis. In this paper, we review briefly the techniques of electrophoresis and illustrate research problems in wildlife and fisheries where the use of electrophoresis is now assisting or may potentially aid in management decisions.

  6. Surface Charge Measurement of SonoVue, Definity and Optison: A Comparison of Laser Doppler Electrophoresis and Micro-Electrophoresis.

    Science.gov (United States)

    Ja'afar, Fairuzeta; Leow, Chee Hau; Garbin, Valeria; Sennoga, Charles A; Tang, Meng-Xing; Seddon, John M

    2015-11-01

    Microbubble (MB) contrast-enhanced ultrasonography is a promising tool for targeted molecular imaging. It is important to determine the MB surface charge accurately as it affects the MB interactions with cell membranes. In this article, we report the surface charge measurement of SonoVue, Definity and Optison. We compare the performance of the widely used laser Doppler electrophoresis with an in-house micro-electrophoresis system. By optically tracking MB electrophoretic velocity in a microchannel, we determined the zeta potentials of MB samples. Using micro-electrophoresis, we obtained zeta potential values for SonoVue, Definity and Optison of -28.3, -4.2 and -9.5 mV, with relative standard deviations of 5%, 48% and 8%, respectively. In comparison, laser Doppler electrophoresis gave -8.7, +0.7 and +15.8 mV with relative standard deviations of 330%, 29,000% and 130%, respectively. We found that the reliability of laser Doppler electrophoresis is compromised by MB buoyancy. Micro-electrophoresis determined zeta potential values with a 10-fold improvement in relative standard deviation.

  7. Effects of multiple polyaniline layers immobilized on carbon nanotube and glutaraldehyde on performance and stability of biofuel cell

    Science.gov (United States)

    Christwardana, Marcelinus; Kwon, Yongchai

    2015-12-01

    Enzymatic biofuel cell (EBC) employing new catalyst for anode electrode is fabricated. The new catalyst consists of glucose oxidase (GOx), polyaniline (PANI) and carbon nanotube (CNT) that are multiply stacked together and finally the stack layer is surrounded by glutaraldehyde (GA) (GA/[GOx/PANI/CNT]n). To evaluate how the GA/[GOx/PANI/CNT]n layer affects EBC performance and stability, electrochemical characterizations are implemented. Regarding optimization, GA/[GOx/PANI/CNT]3 is determined. For elucidating reaction mechanism between glucose and flavin adenine dinucleotide (FAD) of GA/[GOx/PANI/CNT]3, associated investigations are performed. In the evaluations, drop in reduction current peak of FAD is observed with provisions of glucose and O2, while glucose does not influence FAD reaction without O2, confirming O2 makes mediator role. When the GA/[GOx/PANI/CNT]3 layer is adopted, superior catalytic activity and EBC performance are gained (electron transfer rate constant of 5.1 s-1, glucose sensitivity of 150 ìA mM-1 cm-2, and EBC maximum power density (MPD) of 0.29 mW cm-2). Regarding EBC stability, MPD of EBC adopting GA/[GOx/PANI/CNT]3 maintains up to 93% of their initial value even after four weeks. Although GA is little effective for improving EBC performance, EBC stability is helped by GA due to its adhesion promotion capability with [GOx/PANI/CNT]n layer.

  8. Ordered mesoporous carbons obtained by a simple soft template method as sulfur immobilizers for lithium-sulfur cells.

    Science.gov (United States)

    Moreno, Noelia; Caballero, Alvaro; Hernán, Lourdes; Morales, Julián; Canales-Vázquez, Jesús

    2014-08-28

    Carbon materials with ordered mesoporous structures were synthesized using soft template methods and then activated by CO2 treatment. Sulfur was incorporated in these carbons via a simple chemical deposition method in aqueous solutions and the resulting composites were tested as electrodes in Li-S cells. The electrochemical results showed that well-ordered mesoporous carbons perform better than those with a random mesopore arrangement (wormhole-like mesoporous structure). The mesopore ordering yields a framework of well-connected empty sites that results in an enhancement of both the charge carrier mobility and the reversibility of the electrochemical reaction. Although the activation with CO2 partially destroys the mesopore arrangement, which adversely affects the electrode performance, it notably increases the surface area and the micropore content which improves the connectivity between the mesopores. The final observation was an irrelevant effect of the activation process at low current densities. However, at higher rates the activated carbon composite delivered higher capacities. The hierarchical pore structure formed by micro- and mesopores should guarantee the required fast mobility of the Li(+).

  9. A bifunctional poly(ethylene glycol) silane immobilized on metallic oxide-based nanoparticles for conjugation with cell targeting agents

    Energy Technology Data Exchange (ETDEWEB)

    Kohler, Nathan J.; Fryxell, Glen E.; Zhang, Miqin

    2004-06-16

    A trifluoroethylester-terminal poly (ethylene glycol) (PEG) silane was synthesized and self-assembled on iron oxide nanoparticles. The nanoparticle system thus prepared has the flexibility to conjugate with cell targeting agents having either carboxylic and amine terminal groups for a number of biomedical applications, including magnetic resonance imaging (MRI) and controlled drug delivery. The trifluoroethylester silane was synthesized by modifying a PEG diacid to form the corresponding bistrifluoroethylester (TFEE), followed by a reaction with 3-aminopropyltriethoxysilane (APS). The APS coupled with PEG chains confers the stability of PEG self-assembled monolayers (SAMs) and increases the PEG packing density on nanoparticles by establishing hydrogen bonding between the carbonyl and amine groups present within the monolayer structure. The success of the synthesis of the PEG TEFE silane was confirmed with 1H NMR and Fourier transform infrared spectroscopy (FTIR). The conjugating flexibility of the PEG TEFE was demonstrated with folic acid having carboxylic acid groups and amine terminal groups respectively and confirmed by FTIR. TEM analysis showed the dispersion of nanoparticles before and after they were coated with PEG and folic acid.

  10. Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS).

    Science.gov (United States)

    Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A; Nemes, Peter

    2016-08-01

    Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level.

  11. Effect of DNA damage caused by cryopreservation of spermatozoa using a modified Single cell gell electrophoresis in the freshwater catfish Pangasianodon hypophthalmus (Fowler, 1936)

    Institute of Scientific and Technical Information of China (English)

    Kuppusamy Umaa Rani; Natesan Munuswamy

    2014-01-01

    Objective: To detect the integrity of sperm DNA in the catfish Pangasianodon hypophthalmus scirnygolper-ecseelrlv egedl weliethc tHroapnhkosr ebsaisl ainnc oerdd esra ltto stoelsutt ihoonw, 5s%pe-r3m0% c rdyiomperethseyrlv aactieotna mafifdeec t(eDdM nAu) culseianrg D tNhAe stability. Methods: Electrophoresis was conducted for 60 min at 130 mA and 15 V. The comet images awnedre p aenrcaelynzteadge u DsiNnAg isno ftthwea rtaei lC womereet Socbotraein 1e.d5,. aTnhde np tahrea mceotmerest rsautceh a ansd cdoammeatg lee ncgotehf,f ictaieiln lte wngetrhe calculated. Results: There were no significant differences in motility, comet rate and damage coefficient sbpeetwrmee cnr yforepsrhe sseprveermd wainthd 2c5r%yo apnreds 3e0r%ve dD MsAp ehramd sltoowreerd mino t5il%it,y 1, 0h%ig, h1e5r% c oamnde t2 l0e%n gDthM Aa,n dw hdialme athgee coefficients than those of fresh sperm. Conclusions: There was a positive correlation between comet rate of cryopreserved sperm and the concentration of DMA.The toxicity of cryoprotectant is the main factor for DNA damage in cryopreserved sperm.

  12. Characterization and detection of cellular and proteomic alterations in stable stathmin-overexpressing, taxol-resistant BT549 breast cancer cells using offgel IEF/PAGE difference gel electrophoresis.

    Science.gov (United States)

    Balasubramani, Manimalha; Nakao, Chitose; Uechi, Guy T; Cardamone, John; Kamath, Kathy; Leslie, Kristen L; Balachandran, Raghavan; Wilson, Leslie; Day, Billy W; Jordan, Mary Ann

    2011-06-17

    Stathmin/oncoprotein 18, a protein that regulates microtubule dynamics, is highly expressed in a number of tumors including leukemia, lymphoma, neuroblastoma, breast, ovarian, and prostate cancers. High stathmin levels have been associated with the development of resistance to the widely used anti-cancer drug taxol ((®)Taxol, paclitaxel). The mechanisms of stathmin-mediated taxol resistance are not well-understood at the molecular level. To better understand the role of stathmin in taxol resistance, we stably overexpressed stathmin twofold in BT549 human breast cancer cells and characterized several cell processes involved in the mechanism of action of taxol. After stable overexpression of stathmin, neither the cell doubling time nor the mitotic index was altered and the microtubule polymer mass was reduced only modestly (by 18%). Unexpectedly, microtubule dynamicity was reduced by 29% after stathmin overexpression, resulting primarily from reduction in the catastrophe frequency. Sensitivity to taxol was reduced significantly (by 44%) in a clonogenic assay, and stathmin appeared to protect the cells from the spindle-damaging effects of taxol. The results suggest that in the stably stathmin-overexpressing clones, compensatory gene expression occurred that resulted in normal rates of cell proliferation and prevented the increase in catastrophe frequency expected in response to stathmin. Stathmin overexpression protected the cells from taxol-induced abnormal mitoses, and thus induced taxol resistance. Using offgel IEF/PAGE difference gel electrophoresis, we identified a number of proteins whose expression is reduced in the taxol-resistant stathmin-overexpressing cell lines, including proteins involved in the cytoskeleton and cell structure, the stress response, protein folding, glycolysis, and catalysis.

  13. Effect of cell immobilization on the treatment of olive mill wastewater by a total phenols, acetic acid and formic acid degrading bacterium strain

    Directory of Open Access Journals (Sweden)

    Errami, Mohamed

    2005-06-01

    Full Text Available Olive mill wastewater (OMW is a pure vegetative by-product, containing a high organic and polyphenol content and is resistant to biodegradation. Its disposal lead to major environmental pollution problems in the Mediterranean basin. An aerobic bacterium was isolated from OMW. During three consecutive diluted and supplemented OMW treatment cycles, significant abatement of its phytotoxic substances was observed. In fact, total phenols, acetic and formic acids were reduced between 33 and 64 % when cells of the isolated bacterium were grown free; and between 62 and 78 % when cells of the same isolated bacterium were grown immobilized in a polyurethane sponge. These results suggest that the bacterium culture of the new isolate would decrease the OMW phytotoxicity. Phylogenetic analysis of 16S ribosomal DNA showed that all the related sequences are members of the Enterobacteriaceae family and revealed that the isolated bacterium was characterized as a Klebsiella oxytoca strain.El alpechín (OMW es un residuo puro de la extracción del aceite de oliva, que contiene una elevada carga orgánica y de polifenoles por lo que es resistente a la degradación. Su descarga produce graves problemas de contaminación medioambiental en toda el área mediterránea. Se ha aislado una bacteria anaerobia del OMW, que , durante tres ciclos consecutivos de tratamiento del OMW diluido y suplementado, produjo una disminución significativa de las sustancias fitotóxicas del residuo. De hecho, la concentración en fenoles totales, ácido acético y ácido fórmico se redujeron entre 33 y 64 % cuando las células no estaban inmovilizadas y entre el 62 y 78 % cuando las células bacterianas se inmovilizaron en una esponja de poliuretano. Estos resultados indican que el cultivo de la nueva bacteria aislada puede disminuir la fototoxicidad del alpechín. Análisis filogenético del ribosoma 16S de DNA demostró que todas las secuencias eran miembros de la familia

  14. Biodegradation of oil wastewater by free and immobilized Yarrowia lipolytica W29

    Institute of Scientific and Technical Information of China (English)

    WU Lan; GE Gang; WAN Jinbao

    2009-01-01

    The ability of Yarrowia lipolytica W29 immobilized by calcium alginate to degrade oil and chemical oxygen demand (COD) was examined. The degradation rules of oil and COD by immobilized cells with the cell density of 6.65 × 106 CFU/mL degraded 2 000 mg/L oil and 2 000 mg/L COD within 50 h at 30℃ ( pH 7.0, 150 r/min), similarly to those of free cells, and the degradation efficiencies of oil and COD by immobilized cells were above 80%, respectively. At the same time, temperature and initial oil, COD concentration affecting oil and COD degradation of immobilized cells were investigated, the results showed that immobilized cells had high thermostability compared to that of free cells, and substrate concentration significantly affected degrading ability of immobilized cells. Storage stability and reusability tests revealed that the oil degradation ability of immobilized cells was stable after storing at 4℃ for 30 d and reuse for 12 times, respectively, the COD degradation rate of immobilized cells was also maintained 82% at the sixth cycle. These results suggested that immobilized Yarrowia lipolytica might be applicable to a wastewater treatment system for the removal of oil and COD.

  15. Application of capillary electrophoresis in analysis of intact mammalian cells%毛细管电泳在完整哺乳动物细胞分析中的应用

    Institute of Scientific and Technical Information of China (English)

    张璐; 屈锋; 娄蓓蕾

    2012-01-01

    Cell is the basic structural and functional unit of human body. The research of cells' structure, function and behavior is very important. Capillary electrophoresis (CE) is a powerful tool for the separation and analysis, the application of which in cell analysis has progressed significantly. In this paper, the developments of CE applied in the intact mammalian cell analysis are reviewed, which consist of cell population and single cell analysis. The erythrocyte, boar sperm, HeLa cells, SH-SY5Y cells, Caco-2 cells, K562 cells and rat cerebellar granule cells are involved in this review. The methods and conditions for the intact mammalian cell analysis are summarized. In addition, the problems caused by the breakage, aggregation, sedimentation , adsorption and electrophoretic heterogeneity of the cell in the intact mammalian cell analysis by CE are discussed, and the corresponding solutions are introduced. Also, the future research trends are presented. Forty nine papers in all are reviewed.%作为人体形态结构、生理功能和生长发育的基本单位,细胞的结构功能及行为分析具有重要的研究价值.毛细管电泳作为日渐成熟的微量分析技术在细胞分析及应用方面已取得显著进展.本文综述了毛细管电泳在完整哺乳动物细胞分析中的应用,包括群体细胞分析和完整单细胞分析.所述内容涉及血红细胞、公猪精子、人宫颈癌细胞、人神经母细胞瘤细胞、人结直肠腺癌细胞、人慢性髓系白血病细胞以及鼠小脑颗粒细胞.总结了完整哺乳动物细胞分析的毛细管电泳方法和条件,归纳分析了完整细胞分析中存在的细胞破碎、聚集、沉降、吸附及电泳异质性等关键问题及解决方法.对毛细管电泳在细胞分析方面未来的应用方向进行了展望.综述文献49篇.

  16. TO EVALUATE ANAEMIA BY ERYTHROCYTE INDICES, RED CELL DISTRIBUTION WIDTH AND HAEMOGLOBIN ELECTROPHORESIS WITH SPECIAL REFERENCE TO THALASSEMIA IN PAEDIATRIC AGE GROUP

    Directory of Open Access Journals (Sweden)

    Mahendra

    2015-02-01

    Full Text Available Anemia is not a diagnosis; it is a manifestation of an underlying disorder. Thus, even mild, asymptomatic anemia should be investigated so that the primary problem can be diagnosed and treated. Laboratory evaluation begins with a CBC, including WBC and platelet counts, RBC indices and morphology (MCV, MCH, MCHC, RBC distribution width [RDW], and examination of the peripheral smear. In many instances routine test like Hb, TLC, DLC, GBP fail to decide anemia especially in early cases and also fail to decide the type of anaemia. In such situations the RBC indices and RDW are very useful. These become abnormal even before changes in routine hemogram are appreciable. Thalassemia minor poses problems in diagnosis because GBP reveals no features of hemolysis rather it has microcytic hypochromic picture which has similarity with iron deficiency anemia. It is difficult to differentiate between two by only GBP. Several decision making rules have been proposed for differentiation. METHOD : The present study was carried out in 100 cases to evaluate anaemia in different age groups based on RBC Indices and RDW and t o evaluate sensitivity of RBC indices and RDW in diagnosis of anaemia. Cases showing positivity by various rules and RDW in favour of thalassemia minor were subjected to Hb electrophoresis for confirmation of diagnosis. RESULTS : RBC indices are more sensitive for diagnosis of microcytic hypochromic anemia, normocytic normochromic anemia and macrocytic anemia than PBS alone. RDW - CV is superior to all in use to differentiate iron deficiency anemia an d thalassemia minor having high sensitivity 87.3% and specificity 90 .5%.

  17. A large-scale electrophoresis- and chromatography-based determination of gene expression profiles in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties

    Directory of Open Access Journals (Sweden)

    Duban-Deweer Sophie

    2010-11-01

    Full Text Available Abstract Background Brain capillary endothelial cells (BCECs form the physiological basis of the blood-brain barrier (BBB. The barrier function is (at least in part due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein. Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the well-established in vitro BBB model developed in our laboratory. Results A total of 215 protein spots (corresponding to 130 distinct proteins were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin and constitutes valuable evidence for predictions based on genome annotation. Conclusions Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets.

  18. The fluid mechanics of continuous flow electrophoresis

    Science.gov (United States)

    Saville, D. A.

    1990-01-01

    The overall objective is to establish theoretically and confirm experimentally the ultimate capabilities of continuous flow electrophoresis chambers operating in an environment essentially free of particle sedimentation and buoyancy. The efforts are devoted to: (1) studying the effects of particle concentration on sample conductivity and dielectric constant. The dielectric constant and conductivity were identified as playing crucial roles in the behavior of the sample and on the resolving power and throughput of continuous flow devices; and (2) improving the extant mathematical models to predict flow fields and particle trajectories in continuous flow electrophoresis. A dielectric spectrometer was designed and built to measure the complex dielectric constant of a colloidal dispersion as a function of frequency between 500 Hz and 200 kHz. The real part of the signal can be related to the sample's conductivity and the imaginary part to its dielectric constant. Measurements of the dielectric constants of several different dispersions disclosed that the dielectric constants of dilute systems of the sort encountered in particle electrophoresis are much larger than would be expected based on the extant theory. Experiments were carried out to show that, in many cases, this behavior is due to the presence of a filamentary structure of small hairs on the particle surface. A technique for producing electrokinetically ideal synthetic latex particles by heat treating was developed. Given the ubiquitous nature of hairy surfaces with both cells and synthetic particles, it was deemed necessary to develop a theory to explain their behavior. A theory for electrophoretic mobility of hairy particles was developed. Finally, the extant computer programs for predicting the structure of electro-osmotically driven flows were extended to encompass flow channels with variable wall mobilities.

  19. Techniques For Focusing In Zone Electrophoresis

    Science.gov (United States)

    Sharnez, Rizwan; Twitty, Garland E.; Sammons, David W.

    1994-01-01

    In two techniques for focusing in zone electrophoresis, force of applied electrical field in each charged particle balanced by restoring force of electro-osmosis. Two techniques: velocity-gradient focusing (VGF), suitable for rectangular electrophoresis chambers; and field-gradient focusing (FGF), suitable for step-shaped electrophoresis chambers.

  20. Immobilization of Yarrowia lipolytica for aroma production from castor oil.

    Science.gov (United States)

    Braga, Adelaide; Belo, Isabel

    2013-04-01

    The main aim of this study was to compare different materials for Y. lipolytica immobilization that could be used in the production of γ-decalactone (a peach-like aroma) in order to prevent the toxic effect both of the substrate and the aroma upon the cells. Therefore, cells adsorption onto pieces of methyl polymethacrylate and of DupUM(®) was studied and further used in the biotransformation of castor oil into γ-decalactone. The highest aroma concentration was obtained with immobilized cells in DupUM(®), where reconsumption of the aroma by the cells was prevented, contrarily to what happens with free cells. This is a very promising result for γ-decalactone production, with potential to be used at an industrial level since the use of immobilized cells system will facilitate the conversion of a batch process into a continuous mode keeping high cell density and allowing easier recovery of metabolic products.

  1. Immobilization: A Revolution in Traditional Brewing

    Science.gov (United States)

    Virkajärvi, Ilkka; Linko, Matti

    In nature many micro-organisms tend to bind to solid surfaces. This tendency has long been utilized in a number of processes, for example in producing vinegar and acetic acid in bioreactors filled with wood shavings. Acetobacteria are attached to the surface of these shavings. In modern technical language: they are immobilized. Also yeast cells can be immobilized. In the brewing industry this has been the basis for maintaining efficient, continuous fermentation in bioreactors with very high yeast concentrations. The most dramatic change in brewing over recent years has been the replacement of traditional lagering of several weeks by a continuous process in which the residence time is only about 2h. Continuous primary fermentation is used on a commercial scale in New Zealand. In this process, instead of a carrier, yeast is retained in reactors by returning it partly after separation. In many pilot scale experiments the primary fermentation is shortened from about 1week to 1-2days using immobilized yeast reactors. When using certain genetically modified yeast strains no secondary fermentation is needed, and the total fermentation time in immobilized yeast reactors can therefore be shortened to only 2days.

  2. Ratcheted electrophoresis of Brownian particles

    Science.gov (United States)

    Kowalik, Mikołaj; Bishop, Kyle J. M.

    2016-05-01

    The realization of nanoscale machines requires efficient methods by which to rectify unbiased perturbations to perform useful functions in the presence of significant thermal noise. The performance of such Brownian motors often depends sensitively on their operating conditions—in particular, on the relative rates of diffusive and deterministic motions. In this letter, we present a type of Brownian motor that uses contact charge electrophoresis of a colloidal particle within a ratcheted channel to achieve directed transport or perform useful work against an applied load. We analyze the stochastic dynamics of this model ratchet to show that it functions under any operating condition—even in the limit of strong thermal noise and in contrast to existing ratchets. The theoretical results presented here suggest that ratcheted electrophoresis could provide a basis for electrochemically powered, nanoscale machines capable of transport and actuation of nanoscale components.

  3. Capillary electrophoresis systems and methods

    Science.gov (United States)

    Dorairaj, Rathissh; Keynton, Robert S.; Roussel, Thomas J.; Crain, Mark M.; Jackson, Douglas J.; Walsh, Kevin M.; Naber, John F.; Baldwin, Richard P.; Franco, Danielle B.

    2011-08-02

    An embodiment of the invention is directed to a capillary electrophoresis apparatus comprising a plurality of separation micro-channels. A sample loading channel communicates with each of the plurality of separation channels. A driver circuit comprising a plurality of electrodes is configured to induce an electric field across each of the plurality of separation channels sufficient to cause analytes in the samples to migrate along each of the channels. The system further comprises a plurality of detectors configured to detect the analytes.

  4. Capillary electrophoresis in food authenticity.

    Science.gov (United States)

    Kvasnicka, Frantisek

    2005-06-01

    Food authenticity is a term which simply refers to whether the food purchased by the consumer matches its description. False description can occur in many forms, from the undeclared addition of water or other cheaper materials, or the wrong declaration of the amount of a particular ingredient in the product, to making false statements about the source of ingredients i.e., their geographic, plant, or animal origin. The aim of this review is to summarize applications of capillary electrophoresis in food authentication.

  5. Production of ribitol by alginate-immobilized

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, E.C.; Molina, M.C.; Pedrosa, M.M.; Solas, M.T.; vicente, C.; Legaz, M.E. [Laboratorio de Fisiologia, Facultad de Biologia, Madrid (Spain)

    1995-12-01

    Cells of the lichen Cladonia verticillatis have been immobilized in calcium alginate and incubated for 15 days in light on different con centrations of sodium bicarbonate in order to produce lichen polyols. Sugars and polyols secreted to the medium were extracted and analyzed by HPLC. Production and secretion of both ribitol and glucose were very high by entrapped cells incubated on 10 mM sodium bicarbonate but the replacement of calcium by sodium in the alginate matrix produced the disorganization of the immobilized system in a period no longer tan 5 days. however, incubation of the entrapped cells on 1 mM sodium bicarbonate preserved the structure of the innert matrix although only ribitol was produced and secreted to the medium. Entrapment in sodium alginate maintained cell vitality although chloroplast disorganization of the phycobiont became evident. Moreover, contact between myco- and phycobiont has also been altered during immobilization since algal ribitol was not converted into mannitol by the fungal partner. 19 refs.

  6. Capillary Electrophoresis - Optical Detection Systems

    Energy Technology Data Exchange (ETDEWEB)

    Sepaniak, M. J.

    2001-08-06

    Molecular recognition systems are developed via molecular modeling and synthesis to enhance separation performance in capillary electrophoresis and optical detection methods for capillary electrophoresis. The underpinning theme of our work is the rational design and development of molecular recognition systems in chemical separations and analysis. There have been, however, some subtle and exciting shifts in our research paradigm during this period. Specifically, we have moved from mostly separations research to a good balance between separations and spectroscopic detection for separations. This shift is based on our perception that the pressing research challenges and needs in capillary electrophoresis and electrokinetic chromatography relate to the persistent detection and flow rate reproducibility limitations of these techniques (see page 1 of the accompanying Renewal Application for further discussion). In most of our work molecular recognition reagents are employed to provide selectivity and enhance performance. Also, an emerging trend is the use of these reagents with specially-prepared nano-scale materials. Although not part of our DOE BES-supported work, the modeling and synthesis of new receptors has indirectly supported the development of novel microcantilevers-based MEMS for the sensing of vapor and liquid phase analytes. This fortuitous overlap is briefly covered in this report. Several of the more significant publications that have resulted from our work are appended. To facilitate brevity we refer to these publications liberally in this progress report. Reference is also made to very recent work in the Background and Preliminary Studies Section of the Renewal Application.

  7. IMMOBILIZATION OF PAPAIN ON CHITOSAN

    OpenAIRE

    Cahyaningrum, Sari Edi; Narsito, Narsito; Santoso, Sri Juari; Agustini, Rudiana

    2010-01-01

    In this study, papain was immobilized on chitosan with Mg(II) cosslinked agent. Studies on free and immobilized papain systems for determination of optimum pH, optimum temperatur, thermal stability and reusability were carried out. The results showed that free papain had optimum pH 6.5 and optimum temperature 55 °C while the immobile papain hadoptimum pH 8 and optimum temperature 80 °C. The thermal stability of the immobilized papain, relative to that of the free papain, was markedly increase...

  8. Assessment of the ecological security of immobilized enzyme remediation process with biological indicators of soil health.

    Science.gov (United States)

    Zhang, Ying; Dong, Xiaonan; Jiang, Zhao; Cao, Bo; Ge, Shijie; Hu, Miao

    2013-08-01

    This study used the enzymes extracted from an atrazine-degrading strain, Arthrobacter sp. DNS10, which had been immobilized by sodium alginate to rehabilitate atrazine-polluted soil. Meanwhile, a range of biological indices were selected to assess the ecological health of contaminated soils and the ecological security of this bioremediation method. The results showed that there was no atrazine detected in soil samples after 28 days in EN+AT (the soil containing atrazine and immobilized enzyme) treatment. However, the residual atrazine concentration of the sample in AT (the soil containing atrazine only) treatment was about 5.02 ± 0.93 mg kg(-1). These results suggest that the immobilized enzyme exhibits an excellent ability in atrazine degradation. Furthermore, the immobilized enzyme could relieve soil microbial biomass carbon and soil microbial respiration intensity to 772.33 ± 34.93 mg C kg(-1) and 5.01 ± 0.17 mg CO(2) g(-1) soil h(-1), respectively. The results of the polymerase chain reaction-degeneration gradient gel electrophoresis experiment indicated that the immobilized enzyme also could make the Shannon-Wiener index and evenness index of the soil sample increase from 1.02 and 0.74 to 1.51 and 0.84, respectively. These results indicated that the immobilized enzymes not only could relieve the impact from atrazine on the soil, but also revealed that the immobilized enzymes did no significant harm on the soil ecological health.

  9. POTENTIAL APPLICATIONS OF CHITOSAN NANOPARTICLES AS NOVEL SUPPORT IN ENZYME IMMOBILIZATION

    Directory of Open Access Journals (Sweden)

    Hoda Jafarizadeh Malmiri

    2012-01-01

    Full Text Available Chitosan is an attractive natural biopolymer from renewable resources with the presence of reactive amino and hydroxyl functional groups in its structure. Due to the good biocompatibility of chitosan, it can be used in magnetic-field assisted drug delivery, enzyme or cell immobilization and many other industrial applications. In the past decade, nanotechnology has been a considerable research interest in the area of preparation of immobilized enzyme carriers. This study looks at characteristics and applications of chitosan and chitosan nanoparticles and their potentials as suitable supports for enzyme immobilization. Results indicated that activity of immobilized enzymes and performance of enzyme immobilization onto chitosan nanoparticles are higher than chitosan macro and microparticles. As compared to other biopolymers nanoparticles, application of chitosan nanoparticles to immobilize enzymes strongly increases stability of immobilized enzymes and their easy separability from the reaction mixture at the end of the biochemical process.

  10. Tissue stiffness induced by prolonged immobilization of the rat knee joint and relevance of AGEs (pentosidine).

    Science.gov (United States)

    Lee, Sachiko; Sakurai, Takashi; Ohsako, Masafumi; Saura, Ryuichi; Hatta, Hideo; Atomi, Yoriko

    2010-12-01

    Joints, connective tissues consisting of extracellular matrix (ECM) with few blood vessels, transfer tension to the skeleton in response to environmental demand. Therefore, joint immobilization decreases active and passive mechanical stress, resulting in increased joint stiffness and tissue degeneration; however, the cause of joint stiffness is obscure. Using a rat knee immobilization model, we examined the relationship between range of motion (ROM) and cell numbers and ECM cross-links by accumulation of advanced glycation end products, pentosidine, in the posterior joint capsule of immobilized joints during 16 weeks of immobilization. The left knee joint was immobilized by internal fixation and compared with the non-immobilized right leg. As early as 2 weeks of immobilization, joint ROM and torque significantly decreased and in parallel, disordered alignment of collagen fiber bundles significantly increased, compared with non-immobilized joints. Those changes continued until 16 weeks of immobilization. Significant increases in pentosidine-positive areas after 8 weeks and significantly decreased cell numbers after 16 weeks of immobilization were also observed compared to the contralateral side. A significant negative correlation between tissue stiffness measured by restriction of ROM and accumulation of pentosidine was observed. This study is the first to show that immobilization of knee joints induces articular contracture associated with sequential changes of ECM alignment, influencing ROM and later pentosidine accumulation and decreased cell numbers during the 16-week immobilization period. Pentosidine appears to be an indicator toward a chronic tissue stiffness leading to decreased cell number rather than a cause of ROM restriction induced by joint immobilization.

  11. Mapping of Chlamydia trachomatis proteins by immobiline-polyacrylamide two-dimensional electrophoresis: spot identification by N-terminal sequencing and immunoblotting

    DEFF Research Database (Denmark)

    Bini, L; Sanchez-Campillo, M; Santucci, A;

    1996-01-01

    Proteins from purified elementary bodies of Chlamydia trachomatis were separated by two-dimensional gel electrophoresis on nonlinear wide-range immobilized pH gradients in the first dimension and polyacrylamide gradient gels in the second dimension. The maps obtained with this system are highly...

  12. Genotoxicity of three food processing contaminants in transgenic mice expressing human sulfotransferases 1A1 and 1A2 as assessed by the in vivo alkaline single cell gel electrophoresis assay.

    Science.gov (United States)

    Høie, Anja Hortemo; Svendsen, Camilla; Brunborg, Gunnar; Glatt, Hansruedi; Alexander, Jan; Meinl, Walter; Husøy, Trine

    2015-10-01

    The food processing contaminants 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 5-hydroxymethylfurfural (HMF) and 2,5 dimethylfuran (DMF) are potentially both mutagenic and carcinogenic in vitro and/or in vivo, although data on DMF is lacking. The PHIP metabolite N-hydroxy-PhIP and HMF are bioactivated by sulfotransferases (SULTs). The substrate specificity and tissue distribution of SULTs differs between species. A single oral dose of PhIP, HMF or DMF was administered to wild-type (wt) mice and mice expressing human SULT1A1/1A2 (hSULT mice). DNA damage was studied using the in vivo alkaline single cell gel electrophoresis (SCGE) assay. No effects were detected in wt mice. In the hSULT mice, PhIP and HMF exposure increased the levels of DNA damage in the liver and kidney, respectively. DMF was not found to be genotoxic. The observation of increased DNA damage in hSULT mice compared with wt mice supports the role of human SULTs in the bioactivation of N-hydroxy-PhIP and HMF in vivo.

  13. Silica-Immobilized Enzyme Reactors

    Science.gov (United States)

    2007-08-01

    immobilized artificial membrane chromatography and lysophospholipid micellar electrokinetic chromatography . J. Chromatogr. A 1998, 810, 95-103. 50...Journal of Liquid Chromatography and Related Technologies. Air Force Research Laboratory Materials and Manufacturing Directorate Airbase...immobilized enzyme reactors (IMERs) can also be integrated directly to further analytical methods such as liquid chromatography or mass spectrometry.[6] In

  14. Novel absorption detection techniques for capillary electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Yongjun [Iowa State Univ., Ames, IA (United States)

    1994-07-27

    Capillary electrophoresis (CE) has emerged as one of the most versatile separation methods. However, efficient separation is not sufficient unless coupled to adequate detection. The narrow inner diameter (I.D.) of the capillary column raises a big challenge to detection methods. For UV-vis absorption detection, the concentration sensitivity is only at the μM level. Most commercial CE instruments are equipped with incoherent UV-vis lamps. Low-brightness, instability and inefficient coupling of the light source with the capillary limit the further improvement of UV-vis absorption detection in CE. The goals of this research have been to show the utility of laser-based absorption detection. The approaches involve: on-column double-beam laser absorption detection and its application to the detection of small ions and proteins, and absorption detection with the bubble-shaped flow cell.

  15. Capillary electrophoresis theory and practice

    CERN Document Server

    Grossman, Paul D

    1992-01-01

    This book is designed to be a practical guide, used by wide audience, including those new to CE, those more experienced, routine users, those interested in technology development, and those involved with applications research. References have been emphasized to allow the reader to explore the detailed specifics and theoretical foundations.This book draws together the rapidly evolving, diverse, and multidisciplinary subject of capillary electrophoresis (CE). It is designed as a practical guide to be used by a wide audience, including those new to CE as well as more experienced users. T

  16. Electromigration dispersion in Capillary Electrophoresis

    CERN Document Server

    Chen, Zhen; 10.1007/s11538-011-9708-7

    2012-01-01

    In a previous paper (S. Ghosal and Z. Chen Bull. Math. Biol. 2010, vol. 72, pg. 2047) it was shown that the evolution of the solute concentration in capillary electrophoresis is described by a nonlinear wave equation that reduced to Burger's equation if the nonlinearity was weak. It was assumed that only strong electrolytes (fully dissociated) were present. In the present paper it is shown that the same governing equation also describes the situation where the electrolytic buffer consists of a single weak acid (or base). A simple approximate formula is derived for the dimensionless peak variance which is shown to agree well with published experimental data.

  17. An electrochemical method for functionalization of a 316L stainless steel surface being used as a stent in coronary surgery: irreversible immobilization of fibronectin for the enhancement of endothelial cell attachment.

    Science.gov (United States)

    Harvey, Jeffrey; Bergdahl, Andreas; Dadafarin, Hesam; Ling, Li; Davis, Elaine C; Omanovic, Sasha

    2012-06-01

    An electrochemistry-based method for the formation of functionalized alkanethiol layers on a 316L stainless steel surface was developed. The method was efficient in forming a very stable, irreversibly-attached COOH-terminated (mercaptoundecanoic acid) surface layer. This layer was used as a 'linker' to immobilize the extracellular matrix protein fibronectin to the 316L stainless steel surface. Fibronectin was irreversibly attached to the surface and, unlike physisorbed fibronectin, resisted detachment more in aggressive 0.1 M NaOH under sonication. The fibronectin-modified 316L stainless steel surface was more biocompatible towards attachment of endothelial cells than a bare (unmodified) 316L stainless steel surface, yielding a 25% improvement in cell density.

  18. Gene analysis of multiple oral bacteria by the polymerase chain reaction coupled with capillary polymer electrophoresis.

    Science.gov (United States)

    Liu, Chenchen; Yamaguchi, Yoshinori; Sekine, Shinichi; Ni, Yi; Li, Zhenqing; Zhu, Xifang; Dou, Xiaoming

    2016-03-01

    Capillary polymer electrophoresis is identified as a promising technology for the analysis of DNA from bacteria, virus and cell samples. In this paper, we propose an innovative capillary polymer electrophoresis protocol for the quantification of polymerase chain reaction products. The internal standard method was modified and applied to capillary polymer electrophoresis. The precision of our modified internal standard protocol was evaluated by measuring the relative standard deviation of intermediate capillary polymer electrophoresis experiments. Results showed that the relative standard deviation was reduced from 12.4-15.1 to 0.6-2.3%. Linear regression tests were also implemented to validate our protocol. The modified internal standard method showed good linearity and robust properties. Finally, the ease of our method was illustrated by analyzing a real clinical oral sample using a one-run capillary polymer electrophoresis experiment.

  19. Enhanced uranium immobilization and reduction by Geobacter sulfurreducens biofilms.

    Science.gov (United States)

    Cologgi, Dena L; Speers, Allison M; Bullard, Blair A; Kelly, Shelly D; Reguera, Gemma

    2014-11-01

    Biofilms formed by dissimilatory metal reducers are of interest to develop permeable biobarriers for the immobilization of soluble contaminants such as uranium. Here we show that biofilms of the model uranium-reducing bacterium Geobacter sulfurreducens immobilized substantially more U(VI) than planktonic cells and did so for longer periods of time, reductively precipitating it to a mononuclear U(IV) phase involving carbon ligands. The biofilms also tolerated high and otherwise toxic concentrations (up to 5 mM) of uranium, consistent with a respiratory strategy that also protected the cells from uranium toxicity. The enhanced ability of the biofilms to immobilize uranium correlated only partially with the biofilm biomass and thickness and depended greatly on the area of the biofilm exposed to the soluble contaminant. In contrast, uranium reduction depended on the expression of Geobacter conductive pili and, to a lesser extent, on the presence of the c cytochrome OmcZ in the biofilm matrix. The results support a model in which the electroactive biofilm matrix immobilizes and reduces the uranium in the top stratum. This mechanism prevents the permeation and mineralization of uranium in the cell envelope, thereby preserving essential cellular functions and enhancing the catalytic capacity of Geobacter cells to reduce uranium. Hence, the biofilms provide cells with a physically and chemically protected environment for the sustained immobilization and reduction of uranium that is of interest for the development of improved strategies for the in situ bioremediation of environments impacted by uranium contamination.

  20. Bioreporter pseudomonas fluorescens HK44 immobilized in a silica matrix

    Directory of Open Access Journals (Sweden)

    Trogl J.

    2003-01-01

    Full Text Available The bioluminescent bioreporter Pseudomonas fluorescens HK44, the whole cell bacterial biosensor that responds to naphthalene and its metabolites via the production of visible light, was immobilized into a silica matrix by the sol-gel technique. The bioluminescence intensities were measured in the maximum of the bioluminescence band at X = 500 nm. The immobilized cells (>105 cells per g silica matrix produced light after induction by salicylate (cone. > 10 g/l, naphthalene and aminobenzoic acid. The bioluminescence intensities induced by 2,3-dihydroxynaphthalene 3-hydroxybenzoic acid and 4-hydroxybenzoic acid were comparable to a negative control. The cells in the silica layers on glass slides produced light in response to the presence of an inductor at least 8 months after immobilization, and >50 induction cycles. The results showed that these test slides could be used as assays for the multiple determination of water pollution.

  1. Single cell gel electrophoresis as a tool to assess genetic damage in Heleobia cf. australis (Mollusca: Gastropoda as sentinel for industrial and domestic pollution in Montevideo bay (Uruguay

    Directory of Open Access Journals (Sweden)

    Silvia Villar

    2015-09-01

    Full Text Available AbstractThe knowledge of the extent of DNA damage in aquatic organisms in polluted areas is an important issue because contamination may alter their health at sublethal levels. Although molluscs have been widely used to monitor water pollution, there are no records of in vivo genotoxicity studies. Heleobia cf. australis, is distributed in almost all Uruguayan coastal ecosystems, including highly polluted sites. The comet assay is a damage genetic biomarker based on the migration of negatively charged DNA fragments produced by mutagenic agents in individual cells. Live individuals were collected in the Montevideo Bay (impacted area and Laguna Garzón (control to analyze the presence of mutagenic agents in the former site through comet assay. Cells from organisms of the impacted area showed significantly higher levels of genetic damage than those obtained in the control population, measured by percentage of DNA in the tail. Although preliminary, this approach supports the idea that H. cf. australis could be used as a sentinel to evaluate the presence of mutagenic agents in estuarine environments, alerting to the impact of contamination in its early stages.

  2. Degradation of polycyclic aromatic hydrocarbons by free and nanoclay-immobilized manganese peroxidase from Anthracophyllum discolor.

    Science.gov (United States)

    Acevedo, F; Pizzul, L; Castillo, M Dp; González, M E; Cea, M; Gianfreda, L; Diez, M C

    2010-06-01

    Manganese peroxidase (MnP) produced by Anthracophyllum discolor, a Chilean white rot fungus, was immobilized on nanoclay obtained from volcanic soil and its ability to degrade polycyclic aromatic hydrocarbons (PAHs) compared with the free enzyme was evaluated. At the same time, nanoclay characterization was performed. Nanoclay characterization by transmission electronic microscopy showed a particle average size smaller than 100 nm. The isoelectric points (IEP) of nanoclay and MnP from A. discolor were 7.0 and 3.7, respectively, as determined by micro electrophoresis migration and preparative isoelectric focusing. Results indicated that 75% of the enzyme was immobilized on the nanoclay through physical adsorption. As compared to the free enzyme, immobilized MnP from A. discolor achieved an improved stability to temperature and pH. The activation energy (Ea) value for immobilized MnP (51.9 kJ mol(-1)) was higher than that of the free MnP (34.4 kJ mol(-1)). The immobilized enzyme was able to degrade pyrene (>86%), anthracene (>65%), alone or in mixture, and to a less extent fluoranthene (MnP from A. discolor, the enzyme immobilized on nanoclay enhanced the enzymatic transformation of anthracene in soil. Overall results indicate that nanoclay, a carrier of natural origin, is a suitable support material for MnP immobilization. In addition, immobilized MnP shows an increased stability to high temperature, pH and time storage, as well as an enhanced PAHs degradation efficiency in soil. All these characteristics may suggest the possible use of nanoclay-immobilized MnP from A. discolor as a valuable option for in situ bioremediation purposes.

  3. Conducting polymer electrodes for gel electrophoresis.

    Directory of Open Access Journals (Sweden)

    Katarina Bengtsson

    Full Text Available In nearly all cases, electrophoresis in gels is driven via the electrolysis of water at the electrodes, where the process consumes water and produces electrochemical by-products. We have previously demonstrated that π-conjugated polymers such as poly(3,4-ethylenedioxythiophene (PEDOT can be placed between traditional metal electrodes and an electrolyte to mitigate electrolysis in liquid (capillary electroosmosis/electrophoresis systems. In this report, we extend our previous result to gel electrophoresis, and show that electrodes containing PEDOT can be used with a commercial polyacrylamide gel electrophoresis system with minimal impact to the resulting gel image or the ionic transport measured during a separation.

  4. Direct comparison of capillary electrophoresis and capillary liquid chromatography hyphenated to collision-cell inductively coupled plasma mass spectrometry for the investigation of Cd-, Cu- and Zn-containing metalloproteins.

    Science.gov (United States)

    Montes-Bayon, Maria; Pröfrock, Daniel; Sanz-Medel, Alfredo; Prange, Andreas

    2006-05-05

    Capillary liquid chromatography (cLC) and capillary electrophoresis (CE) have been critically compared for the separation of metalloproteins when using collision-cell inductively coupled plasma mass spectrometry (ICP-CC-MS) as detection system. For cLC separation, the selected column was a C8 (0.3 mm I.D.) and the separation conditions involved a gradient up to 80% methanol in 10mM ammonium acetate buffer (pH 7.4). The low flow rate used (3 microL min(-1)) permitted the utilization of a high methanol content maintaining the sensitivity along the whole chromatographic run. For this purpose, a new low-flow interface has been developed based on a total consumption nebulizer. Similarly, CE has been studied as separation technique using a 75 microm I.D. fused silica capillary and a running buffer of 20 mM Tris-HNO3 (pH 7.4) and working at 30 kV. Metallothionein (mixture of MT-I and -II) and superoxide dismutase (SOD) have been used as protein models in order to evaluate the separation/detection capabilities using the same injection volumes in both systems (20 nL). For both hybrid systems, separation parameters such as retention factor, numbers of theoretical plates, tailing factor and resolution have been critically compared. Also, the analytical performance characteristics of both hybrid systems have been evaluated and tested by analyzing the Cu-, Zn-species present in red blood cell extracts in order to explore more adequate separation methodology for the analysis of metalloproteins in complex matrices.

  5. Colonization of Bordetella pertussis clinical isolates that differ by pulsed field gel electrophoresis types in the lungs of naïve mice or mice immunized with the whole-cell pertussis vaccine used in Poland.

    Science.gov (United States)

    Polak, Maciej; Zawadka, Monika; Mosiej, Ewa; Rabczenko, Daniel; Augustynowicz, Ewa; Guiso, Nicole; Lutyńska, Anna

    2015-04-01

    The goal of our study was to compare the elimination of Bordetella pertussis clinical isolates that differ according to pulsed field gel electrophoresis (PFGE), serotypes and genes encoding virulence factors from the lungs of naïve mice or mice immunized with commercial diphtheria-tetanus-whole-cell pertussis vaccine used in Poland. When a mixture of four isolates, given in equal proportions and harboring different PFGE profiles, serotypes, and alleles encoding virulence factors, was used to infect non-immunized mice, a single isolate, characterized by PFGE type IVγ, Fim2 phenotype and ptxA1-prn2-tcfA2-fim2-1-ptxP1-ptxC1-fim3-1 alleles, was found to be significantly predominant compared to the others. This PFGE profile is commonly found in B. pertussis isolates circulating in some European countries since the late 1990s, confirming its high fitness. The Polish commercial whole-cell pertussis vaccine induced an immunity effective at eliminating the B. pertussis isolates from the lungs. However, the elimination of the isolate harboring PFGE type C profile, Fim2,3 phenotype and ptxA1-prn1-tcfA2-fim2-1-ptxP1-ptxC1-fim3-1 alleles was delayed as compared to the others, suggesting phenotypic differences with the other isolates and vaccine strains. Nevertheless, the same isolate, when challenged into mice in the defined mixture of strains, lost the competition with the others, as measured by lung colonization efficiency. This PFGE profile represents 15 % of the isolates circulating in Poland between 2001 and 2012.

  6. DNA Sequencing Using capillary Electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Barry Karger

    2011-05-09

    The overall goal of this program was to develop capillary electrophoresis as the tool to be used to sequence for the first time the Human Genome. Our program was part of the Human Genome Project. In this work, we were highly successful and the replaceable polymer we developed, linear polyacrylamide, was used by the DOE sequencing lab in California to sequence a significant portion of the human genome using the MegaBase multiple capillary array electrophoresis instrument. In this final report, we summarize our efforts and success. We began our work by separating by capillary electrophoresis double strand oligonucleotides using cross-linked polyacrylamide gels in fused silica capillaries. This work showed the potential of the methodology. However, preparation of such cross-linked gel capillaries was difficult with poor reproducibility, and even more important, the columns were not very stable. We improved stability by using non-cross linked linear polyacrylamide. Here, the entangled linear chains could move when osmotic pressure (e.g. sample injection) was imposed on the polymer matrix. This relaxation of the polymer dissipated the stress in the column. Our next advance was to use significantly lower concentrations of the linear polyacrylamide that the polymer could be automatically blown out after each run and replaced with fresh linear polymer solution. In this way, a new column was available for each analytical run. Finally, while testing many linear polymers, we selected linear polyacrylamide as the best matrix as it was the most hydrophilic polymer available. Under our DOE program, we demonstrated initially the success of the linear polyacrylamide to separate double strand DNA. We note that the method is used even today to assay purity of double stranded DNA fragments. Our focus, of course, was on the separation of single stranded DNA for sequencing purposes. In one paper, we demonstrated the success of our approach in sequencing up to 500 bases. Other

  7. Kinetic Measurements for Enzyme Immobilization.

    Science.gov (United States)

    Cooney, Michael J

    2017-01-01

    Enzyme kinetics is the study of the chemical reactions that are catalyzed by enzymes, with a focus on their reaction rates. The study of an enzyme's kinetics considers the various stages of activity, reveals the catalytic mechanism of this enzyme, correlates its value to assay conditions, and describes how a drug or a poison might inhibit the enzyme. Victor Henri initially reported that enzyme reactions were initiated by a bond between the enzyme and the substrate. By 1910, Michaelis and Menten were advancing their work by studying the kinetics of an enzyme saccharase which catalyzes the hydrolysis of sucrose into glucose and fructose. They published their analysis and ever since the Michaelis-Menten equation has been used as the standard to describe the kinetics of many enzymes. Unfortunately, soluble enzymes must generally be immobilized to be reused for long times in industrial reactors. In addition, other critical enzyme properties have to be improved like stability, activity, inhibition by reaction products, and selectivity towards nonnatural substrates. Immobilization is by far the chosen process to achieve these goals.Although the Michaelis-Menten approach has been regularly adapted to the analysis of immobilized enzyme activity, its applicability to the immobilized state is limited by the barriers the immobilization matrix places upon the measurement of compounds that are used to model enzyme kinetics. That being said, the estimated value of the Michaelis-Menten coefficients (e.g., V max, K M) can be used to evaluate effects of immobilization on enzyme activity in the immobilized state when applied in a controlled manner. In this review enzyme activity and kinetics are discussed in the context of the immobilized state, and a few novel protocols are presented that address some of the unique constraints imposed by the immobilization barrier.

  8. Immobilization and catalytic properties of lipase on chitosan for hydrolysis and esterification reactions

    OpenAIRE

    2003-01-01

    The objective of this study was to evaluate the immobilization of lipase on a chitosan support by physical adsorption, aiming at its application in hydrolytic and synthetic reactions. Two types of chitosan (flakes and porous) were used for immobilizing lipase from a microbial source (Candida rugosa) and animal cells (porcine pancreas). The best results for recovery of total activity after immobilization were obtained for microbial lipase and porous chitosan beads. This set was selected for fu...

  9. Electrophoresis tests on STS-3 and ground control experiments - A basis for future biological sample selections

    Science.gov (United States)

    Morrison, D. R.; Lewis, M. L.

    1982-01-01

    Static zone electrophoresis is an electrokinetic method of separating macromolecules and small particles. However, its application for the isolation of biological cells and concentrated protein solutions is limited by sedimentation and convection. Microgravity eliminates or reduces sedimentation, floatation, and density-driven convection arising from either Joule heating or concentration differences. The advantages of such an environment were first demonstrated in space during the Apollo 14 and 16 missions. In 1975 the Electrophoresis Technology Experiment (MA-011) was conducted during the Apollo-Soyuz Test Project flight. In 1979 a project was initiated to repeat the separations of human kidney cells. One of the major objectives of the Electrophoresis Equipment Verification Tests (EEVT) on STS-3 was to repeat and thereby validate the first successful electrophoretic separation of human kidney cells. Attention is given to the EEVT apparatus, the preflight electrophoresis, and inflight operational results.

  10. Proteome analysis of vaccinia virus IHD-W-infected HEK 293 cells with 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS of on solid phase support N-terminally sulfonated peptides

    Directory of Open Access Journals (Sweden)

    Bartel Sebastian

    2011-08-01

    Full Text Available Abstract Background Despite the successful eradication of smallpox by the WHO-led vaccination programme, pox virus infections remain a considerable health threat. The possible use of smallpox as a bioterrorism agent as well as the continuous occurrence of zoonotic pox virus infections document the relevance to deepen the understanding for virus host interactions. Since the permissiveness of pox infections is independent of hosts surface receptors, but correlates with the ability of the virus to infiltrate the antiviral host response, it directly depends on the hosts proteome set. In this report the proteome of HEK293 cells infected with Vaccinia Virus strain IHD-W was analyzed by 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS in a bottom-up approach. Results The cellular and viral proteomes of VACV IHD-W infected HEK293 cells, UV-inactivated VACV IHD-W-treated as well as non-infected cells were compared. Derivatization of peptides with 4-sulfophenyl isothiocyanate (SPITC carried out on ZipTipμ-C18 columns enabled protein identification via the peptides' primary sequence, providing improved s/n ratios as well as signal intensities of the PSD spectra. The expression of more than 24 human proteins was modulated by the viral infection. Effects of UV-inactivated and infectious viruses on the hosts' proteome concerning energy metabolism and proteins associated with gene expression and protein-biosynthesis were quite similar. These effects might therefore be attributed to virus entry and virion proteins. However, the modulation of proteins involved in apoptosis was clearly correlated to infectious viruses. Conclusions The proteome analysis of infected cells provides insight into apoptosis modulation, regulation of cellular gene expression and the regulation of energy metabolism. The confidence of protein identifications was clearly improved by the peptides' derivatization with SPITC on a solid phase support. Some of the identified proteins

  11. Electrophoresis-mass spectrometry probe

    Science.gov (United States)

    Andresen, Brian D.; Fought, Eric R.

    1987-01-01

    The invention involves a new technique for the separation of complex mixtures of chemicals, which utilizes a unique interface probe for conventional mass spectrometers which allows the electrophoretically separated compounds to be analyzed in real-time by a mass spectrometer. This new chemical analysis interface, which couples electrophoresis with mass spectrometry, allows complex mixtures to be analyzed very rapidly, with much greater specificity, and with greater sensitivity. The interface or probe provides a means whereby large and/or polar molecules in complex mixtures to be completely characterized. The preferred embodiment of the probe utilizes a double capillary tip which allows the probe tip to be continually wetted by the buffer, which provides for increased heat dissipation, and results in a continually operating interface which is more durable and electronically stable than the illustrated single capillary tip probe interface.

  12. Non-Aqueous Capillary Electrophoresis

    Science.gov (United States)

    Szumski, Michał; Buszewski, Bogusław

    Non-aqueous capillary electrophoresis and capillary electrochromatography are special variants of these techniques. Here, organic solvents or their mixtures with or without dissolved electrolytes are used as separation buffer or mobile phase, respectively. The most important features of non-aqueous systems are: better solubility of more hydrophobic ionic substances (many natural products) than in water, much less current and Joule heating allows for using highly concentrated buffers and/or larger capillary internal diameters, polar interactions are enhanced in organic solvents which is often highly advantageous in chiral separation systems. This chapter presents most frequently used solvents, their properties, as well as shows pH* scale which is often used in non-aqueous systems.

  13. IMMOBILIZATION OF LYSOZYME IN POLYVINYL ALCOHOL CRYOGEL

    Directory of Open Access Journals (Sweden)

    S. S. Dekina

    2014-06-01

    Full Text Available The lysozyme immobilization in cryogel of polyvinyl alcohol and physico-chemical properties of obtained preparation was investigated. Hydrolytic activity of lysozyme was determined by bacteriolytic method, using Micrococcus lysodeikticus cells acetone powder as substrate. Protein content was determined by the Lowry–Hartree method. Immobilization of lysozyme was conducted by entrapment in polyvinyl alcohol gel with subsequent cycles of freezing-thawing. Antimicrobial activity was studied by standard disk-diffusional method. The hydrogel filmic coatings with antimicrobial action, insoluble at physiological conditions, with quantitative retaining of protein and hydrolytic activity of lysozyme were obtained. The product is characterized by the widened pH-profile of activity at acidic pH values, stability in acidic medium (pH 5.5 and at storage. Its antimicrobial action against Staphylococcus aureus ATCC 25923 F-49, Pseudomonas aeruginosa 415, Escherichia coli 055 K 59912/4 and Candida albicans ATCC 885-653 was noted. The proposed method of lysozyme immobilization allows to obtain stable, highly effective product with antimicrobial activity, prospective for usage in biomedical investigations.

  14. Enhanced butanol production by immobilized Clostridium beijerinckii TISTR 1461 using zeolite 13X as a carrier.

    Science.gov (United States)

    Vichuviwat, Rapeephat; Boonsombuti, Akarin; Luengnaruemitchai, Apanee; Wongkasemjit, Sujitra

    2014-11-01

    Butanol production by cell immobilization onto porous materials-brick and zeolite 13X-was investigated using Clostridium beijerinckii TISTR 1461. Characterization results of two materials were completed to evaluate their potential as an immobilization carrier. Although zeolite has greater porosity than brick, it cannot be used for cell aggregation without treating with chemical. After immobilization, both materials can enhance butanol titers from 5.29 to 5.80g/L and 8.58g/L using brick and zeolite, respectively. Butanol to glucose yield also improved from 0.14 to 0.16g/g after immobilization. It was found that butanol production significantly increased due to an increase in buffering capacity, strong bonding between the zeolite surface and cell, and butanol tolerance. In addition, repeated batch fermentation was performed, demonstrating that cells immobilized onto zeolite 13X have high stability and potential for long-term use in continuous fermentation.

  15. Compensating for Electro-Osmosis in Electrophoresis

    Science.gov (United States)

    Rhodes, Percy H.; Snyder, Robert S.

    1987-01-01

    Simple mechanical adjustment eliminates transverse velocity component. New apparatus for moving-wall electrophoresis increases degree of collimation of chemical species in sample stream. Electrophoresis chamber set at slight angle in horizontal plane to adjust angle between solution flow and wall motion. Component of velocity created cancels electro-osmotic effect.

  16. Treating Wastewater With Immobilized Enzymes

    Science.gov (United States)

    Jolly, Clifford D.

    1991-01-01

    Experiments show enzymes are immobilized on supporting materials to make biocatalyst beds for treatment of wastewater. With suitable combination of enzymes, concentrations of various inorganic and organic contaminants, including ammonia and urea, reduced significantly.

  17. Ultrafast Capillary Electrophoresis Isolation of DNA Aptamer for the PCR Amplification-Based Small Analyte Sensing

    Science.gov (United States)

    Fiore, Emmanuelle; Dausse, Eric; Dubouchaud, Hervé; Peyrin, Eric; Ravelet, Corinne

    2015-08-01

    Here, we report a new homogeneous DNA amplification-based aptamer assay for small analyte sensing. The aptamer of adenosine chosen as the model analyte was split into two fragments able to assemble in the presence of target. Primers were introduced at extremities of one fragment in order to generate the amplifiable DNA component. The amount of amplifiable fragment was quantifiable by Real-Time Polymerase Chain Reaction (RT-PCR) amplification and directly reliable on adenosine concentration. This approach combines the very high separation efficiency and the homogeneous format (without immobilization) of capillary electrophoresis and the sensitivity of real time PCR amplification. An ultrafast isolation of target-bound split aptamer (60 s) was developed by designing a capillary electrophoresis input/ouput scheme. Such method was successfully applied to the determination of adenosine with a LOD of 1 µM.

  18. Aerobic degradation of nitrobenzene by immobilization of Rhodotorula mucilaginosa in polyurethane foam.

    Science.gov (United States)

    Zheng, Chunli; Zhou, Jiti; Wang, Jing; Qu, Baocheng; Wang, Jing; Lu, Hong; Zhao, Hongxia

    2009-08-30

    Rhodotorula mucilaginosa Z1 capable of degrading nitrobenzene was immobilized in polyurethane foam. The nitrobenzene-degrading capacity of immobilized cells was compared to free cells in batches in shaken culture. Effects of pH and temperature on the nitrobenzene degradation showed that polyurethane-immobilized Z1 had higher tolerances toward acid, alkali, and heat than those of free cells. Kinetic studies revealed that higher concentrations of nitrobenzene were better tolerated and more quickly degraded by polyurethane-immobilized Z1 than by free cells. Moreover, the ability of polyurethane-immobilized Z1 to resist nitrobenzene shock load was enhanced. Experiments on the nitrobenzene degradation in different concentrations of NaCl and in the presence of phenol or aniline demonstrated that polyurethane-immobilized Z1 exhibited higher tolerance toward salinity and toxic chemicals than those of free cells. Immobilization therefore could be a promising method for treating nitrobenzene industrial wastewater. This is the first report on the degradation of nitrobenzene by a polyurethane-immobilized yeast strain.

  19. Immobilization of cellulases on magnetic particles to enable enzyme recycling during hydrolysis of lignocellulose

    DEFF Research Database (Denmark)

    Alftrén, Johan

    on commercial magnetic particles coated with streptavidin. The procedure enabled simultaneous purification and immobilization from crude cell lysate because of the very strong interaction and high affinity between biotin and streptavidin. A third method of immobilizing enzymes was employed in paper IV where two...... feedstocks containing insolubles. This could potentially be overcome by immobilizing the cellulases on magnetically susceptible particles. Consequently, the immobilized cellulases could be magnetically recovered and recycled for a new cycle of enzymatic hydrolysis of cellulose. The main objective...... of this thesis was to examine the possibility of immobilizing cellulases on magnetic particles in order to enable enzyme re-use. Studies at lab and pilot scale (20 L) were conducted using model and real substrates. In paper I and III beta-glucosidase or a whole cellulase mixture was covalently immobilized...

  20. Proteomic Comparison of Two-Dimensional Gel Electrophoresis Pro files from Human Lung Squamous Carcinoma and Normal Bronchial Epithelial Tissues

    Institute of Scientific and Technical Information of China (English)

    Cui Li; Ping Chen; Jingyun Xie; Songping Liang; Xianquan Zhan; Maoyu Li; Xiaoying Wu; Feng Li; Jianling Li; Zhiqiang Xiao; Zhuchu Chen; Xueping Feng

    2003-01-01

    Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-ug protein-load. The average deviation of spot position was 0.733+0.101 mm in IEF direction, and 0.925+0.207 mm in SDS-PAGE direction. For tumor tissue, a total of 1241±88 spots were detected, 987±65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190+72 spots were detected, and 875±48 spots were matched with an average matching rate of 73.5%. A total of 864±34 spots were matched between tumors and controls.Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduction. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis.These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.

  1. Poly(Dopamine-Assisted Immobilization of Xu Duan on 3D Printed Poly(Lactic Acid Scaffolds to Up-Regulate Osteogenic and Angiogenic Markers of Bone Marrow Stem Cells

    Directory of Open Access Journals (Sweden)

    Chia-Hung Yeh

    2015-07-01

    Full Text Available Three-dimensional printing is a versatile technique to generate large quantities of a wide variety of shapes and sizes of polymer. The aim of this study is to develop functionalized 3D printed poly(lactic acid (PLA scaffolds and use a mussel-inspired surface coating and Xu Duan (XD immobilization to regulate cell adhesion, proliferation and differentiation of human bone-marrow mesenchymal stem cells (hBMSCs. We prepared PLA scaffolds and coated with polydopamine (PDA. The chemical composition and surface properties of PLA/PDA/XD were characterized by XPS. PLA/PDA/XD controlled hBMSCs’ responses in several ways. Firstly, adhesion and proliferation of hBMSCs cultured on PLA/PDA/XD were significantly enhanced relative to those on PLA. In addition, the focal adhesion kinase (FAK expression of cells was increased and promoted cell attachment depended on the XD content. In osteogenesis assay, the osteogenesis markers of hBMSCs cultured on PLA/PDA/XD were significantly higher than seen in those cultured on a pure PLA/PDA scaffolds. Moreover, hBMSCs cultured on PLA/PDA/XD showed up-regulation of the ang-1 and vWF proteins associated with angiogenic differentiation. Our results demonstrate that the bio-inspired coating synthetic PLA polymer can be used as a simple technique to render the surfaces of synthetic scaffolds active, thus enabling them to direct the specific responses of hBMSCs.

  2. Non-invasive screening for Alzheimer's disease by sensing salivary sugar using Drosophila cells expressing gustatory receptor (Gr5a) immobilized on an extended gate ion-sensitive field-effect transistor (EG-ISFET) biosensor.

    Science.gov (United States)

    Lau, Hui-Chong; Lee, In-Kyu; Ko, Pan-Woo; Lee, Ho-Won; Huh, Jeung-Soo; Cho, Won-Ju; Lim, Jeong-Ok

    2015-01-01

    Body fluids are often used as specimens for medical diagnosis. With the advent of advanced analytical techniques in biotechnology, the diagnostic potential of saliva has been the focus of many studies. We recently reported the presence of excess salivary sugars, in patients with Alzheimer's disease (AD). In the present study, we developed a highly sensitive, cell-based biosensor to detect trehalose levels in patient saliva. The developed biosensor relies on the overexpression of sugar sensitive gustatory receptors (Gr5a) in Drosophila cells to detect the salivary trehalose. The cell-based biosensor was built on the foundation of an improved extended gate ion-sensitive field-effect transistor (EG-ISFET). Using an EG-ISFET, instead of a traditional ion-sensitive field-effect transistor (ISFET), resulted in an increase in the sensitivity and reliability of detection. The biosensor was designed with the gate terminals segregated from the conventional ISFET device. This design allows the construction of an independent reference and sensing region for simultaneous and accurate measurements of samples from controls and patients respectively. To investigate the efficacy of the cell-based biosensor for AD screening, we collected 20 saliva samples from each of the following groups: participants diagnosed with AD, participants diagnosed with Parkinson's disease (PD), and a control group composed of healthy individuals. We then studied the response generated from the interaction of the salivary trehalose of the saliva samples and the Gr5a in the immobilized cells on an EG-ISFET sensor. The cell-based biosensor significantly distinguished salivary sugar, trehalose of the AD group from the PD and control groups. Based on these findings, we propose that salivary trehalose, might be a potential biomarker for AD and could be detected using our cell-based EG-ISFET biosensor. The cell-based EG-ISFET biosensor provides a sensitive and direct approach for salivary sugar detection and

  3. DNA Sequencing Using capillary Electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Barry Karger

    2011-05-09

    The overall goal of this program was to develop capillary electrophoresis as the tool to be used to sequence for the first time the Human Genome. Our program was part of the Human Genome Project. In this work, we were highly successful and the replaceable polymer we developed, linear polyacrylamide, was used by the DOE sequencing lab in California to sequence a significant portion of the human genome using the MegaBase multiple capillary array electrophoresis instrument. In this final report, we summarize our efforts and success. We began our work by separating by capillary electrophoresis double strand oligonucleotides using cross-linked polyacrylamide gels in fused silica capillaries. This work showed the potential of the methodology. However, preparation of such cross-linked gel capillaries was difficult with poor reproducibility, and even more important, the columns were not very stable. We improved stability by using non-cross linked linear polyacrylamide. Here, the entangled linear chains could move when osmotic pressure (e.g. sample injection) was imposed on the polymer matrix. This relaxation of the polymer dissipated the stress in the column. Our next advance was to use significantly lower concentrations of the linear polyacrylamide that the polymer could be automatically blown out after each run and replaced with fresh linear polymer solution. In this way, a new column was available for each analytical run. Finally, while testing many linear polymers, we selected linear polyacrylamide as the best matrix as it was the most hydrophilic polymer available. Under our DOE program, we demonstrated initially the success of the linear polyacrylamide to separate double strand DNA. We note that the method is used even today to assay purity of double stranded DNA fragments. Our focus, of course, was on the separation of single stranded DNA for sequencing purposes. In one paper, we demonstrated the success of our approach in sequencing up to 500 bases. Other

  4. Immobilization of Streptomyces thermotolerans 11432 on polyurethane foam to improve production of acetylisovaleryltylosin.

    Science.gov (United States)

    Zhu, Hongji; Wang, Weihua; Liu, Jiaheng; Caiyin, Qinggele; Qiao, Jianjun

    2015-01-01

    In this study, polyurethane foam (PUF) was chemically treated to immobilize Streptomyces thermotolerans 11432 for semi-continuous production of acetylisovaleryltylosin (AIV). Based on experimental results, positive cross-linked PUF (PCPUF) was selected as the most effective carrier according to immobilized cell mass. The effect of adsorption time on immobilized mass was investigated. AIV concentration (33.54 mg/l) in batch fermentations with immobilized cells was higher than with free cells (20.34 mg/l). In repeated batch fermentations with immobilized S. thermotolerans 11432 using PCPUF cubes, high AIV concentrations and conversion rates were attained, ranging from 25.56 to 34.37 mg/l and 79.93 to 86.31 %, respectively. Significantly, this method provides a feasible strategy for efficient AIV production and offers the potential for large-scale production.

  5. Status of plutonium ceramic immobilization processes and immobilization forms

    Energy Technology Data Exchange (ETDEWEB)

    Ebbinghaus, B.B.; Van Konynenburg, R.A. [Lawrence Livermore National Lab., CA (United States); Vance, E.R.; Jostsons, A. [Australian Nuclear Science and Technology Organization, Menai (Australia)] [and others

    1996-05-01

    Immobilization in a ceramic followed by permanent emplacement in a repository or borehole is one of the alternatives currently being considered by the Fissile Materials Disposition Program for the ultimate disposal of excess weapons-grade plutonium. To make Pu recovery more difficult, radioactive cesium may also be incorporated into the immobilization form. Valuable data are already available for ceramics form R&D efforts to immobilize high-level and mixed wastes. Ceramics have a high capacity for actinides, cesium, and some neutron absorbers. A unique characteristic of ceramics is the existence of mineral analogues found in nature that have demonstrated actinide immobilization over geologic time periods. The ceramic form currently being considered for plutonium disposition is a synthetic rock (SYNROC) material composed primarily of zirconolite (CaZrTi{sub 2}O{sub 7}), the desired actinide host phase, with lesser amounts of hollandite (BaAl{sub 2}Ti{sub 6}O{sub 16}) and rutile (TiO{sub 2}). Alternative actinide host phases are also being considered. These include pyrochlore (Gd{sub 2}Ti{sub 2}O{sub 7}), zircon (ZrSiO{sub 4}), and monazite (CePO{sub 4}), to name a few of the most promising. R&D activities to address important technical issues are discussed. Primarily these include moderate scale hot press fabrications with plutonium, direct loading of PuO{sub 2} powder, cold press and sinter fabrication methods, and immobilization form formulation issues.

  6. Covalent co-immobilization of heparin/laminin complex that with different concentration ratio on titanium surface for selectively direction of platelets and vascular cells behavior

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jian; Chen, Yuan; Liu, Tao; Wang, Xue; Liu, Yang; Wang, Yuan; Chen, Junying, E-mail: chenjy@263.net; Huang, Nan

    2014-10-30

    Highlights: • Extracellular matrix inspired surface modification with fibronectin, heparin and VEGF to construct a favorable microenvironment for selectively anticoagulant and promote endothelialization. • Take the advantage of specific intermolecular interaction, the bioactivity of above biomolecules was more efficiently maintained in compared with the common used covalent immobilization method. • Poly-l-lysine was used as a novel interlayer for surface amination, and in comparison, PLL coating was more feasible and the degradation product had no harm to human body. - Abstract: Surface biofunctional modification of coronary artery stent to improve the hemocompatibility and selectively accelerate endothelium regeneration but prevent restenosis have been become a new hotspot. For this, a novel method was developed in this work by co-immobilization of Ln and heparin complex on poly-L-lysine modified Ti surface. Take the advantage of the specific interaction between Ln and heparin, Ln and heparin complexes with different concentration ratios were set up for creating different exposure density of these two types of biomolecules. According to biocompatibility evaluation results, the Hep/Ln complexes modified surface displayed less platelet adhesion and activation. Especially, on L(150)H and L(200)H surface, the AT III binding quantity, APTT value and anti-coagulation property of modified surface were significantly promoted. Furthermore, the adherent density and proliferation activity of ECs and EPCs were positively correlated with Ln concentration. Notably, the proliferation of both ECs and EPCs on L(100)H, L(150)H and L(200)H surface were greatly promoted. Another hand, the proliferation activity of SMCs was significantly inhibited on Hep/Ln modified surfaces, which was considered mainly due to the inhibitory effect of heparin to SMCs. According to the existing results, this study demonstrated that in a certain range of heparin and laminin concentration ratio

  7. Cobalt immobilization by manganese oxidizing bacteria from the Indian ridge system

    Digital Repository Service at National Institute of Oceanography (India)

    Antony, R.; Sujith, P.P.; Fernandes, S.O.; Verma, P.; Khedekar, V.D.; LokaBharathi, P.A.

    Co immobilization by two manganese oxidizing isolates from Carlsberg Ridge waters (CR35 and CR48) was compared with that of Mn at same molar concentrations. At a lower concentration of 10 mu M, CR35 and CR48 immobilized 22 and 23 fM Co cell-1...

  8. Phosphopeptide enrichment by immobilized metal affinity chromatography

    DEFF Research Database (Denmark)

    Thingholm, Tine E.; Larsen, Martin R.

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively...... charged metal ions such as Fe3+, Ga3+, Al3+, Zr4+, and Ti4+ has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from...

  9. Characterization of the dynamic behavior of nonlinear biosystems in the presence of model uncertainty using singular invariance PDEs: application to immobilized enzyme and cell bioreactors.

    Science.gov (United States)

    Kazantzis, Nikolaos; Kazantzi, Vasiliki

    2010-04-01

    A new approach to the problem of characterizing the dynamic behavior of nonlinear biosystems in the presence of model uncertainty using the notion of slow invariant manifold is proposed. The problem of interest is addressed within the context of singular partial differential equations (PDE) theory, and in particular, through a system of singular quasi-linear invariance PDEs for which a general set of conditions for solvability is provided. Within the class of analytic solutions, this set of conditions guarantees the existence and uniqueness of a locally analytic solution which represents the system's slow invariant manifold exponentially attracting all dynamic trajectories in the absence of model uncertainty. An exact reduced-order model is then obtained through the restriction of the original biosystem dynamics on the slow manifold. The analyticity property of the solution to the invariance PDEs enables the development of a series solution method that can be easily implemented using MAPLE leading to polynomial approximations up to the desired degree of accuracy. Furthermore, the aforementioned attractivity property and the transition towards the above manifold is analyzed and characterized in the presence of model uncertainty. Finally, examples of certain immobilized enzyme bioreactors are considered to elucidate aspects of the proposed context of analysis.

  10. Atomic Force Controlled Capillary Electrophoresis

    Science.gov (United States)

    Lewis, Aaron; Yeshua, Talia; Palchan, Mila; Lovsky, Yulia; Taha, Hesham

    2010-03-01

    Lithography based on scanning probe microscopic techniques has considerable potential for accurate & localized deposition of material on the nanometer scale. Controlled deposition of metallic features with high purity and spatial accuracy is of great interest for circuit edit applications in the semiconductor industry, for plasmonics & nanophotonics and for basic research in surface enhanced Raman scattering & nanobiophysics. Within the context of metal deposition we will review the development of fountain pen nanochemistry and its most recent emulation Atomic Force Controlled Capillary Electrophoresis (ACCE). Using this latter development we will demonstrate achievement of unprecedented control of nanoparticle deposition using a three-electrode geometry. Three electrodes are attached: one on the outside of a metal coated glass probe, one on the inside of a hollow probe in a solution containing Au nanoparticles in the capillary, and a third on the surface where the writing takes place. The three electrodes provide electrical pulses for accurate control of deposition and retraction of the liquid from the surface overcoming the lack of control seen in both dip pen lithography & fountain pen nanochemistry when the tip contacts the surface. With this development, we demonstrate depositing a single 1.3 nm Au nanoparticle onto surfaces such as semiconductors.

  11. [Disc electrophoresis of collagen protein (author's transl)].

    Science.gov (United States)

    Reitmayr, P; Verzár, F

    1975-01-01

    The composition of proteins extracted from tendon collagen is investigated by disc electrophoresis. No qualitative differences can be demonstrated between young and old collagen. The action of formaldehyde and methionine on the tendons has no effect on the electrophoretic picture.

  12. Immobile Complex Verbs in Germanic

    DEFF Research Database (Denmark)

    Vikner, Sten

    2005-01-01

    Certain complex verbs in Dutch, German, and Swiss German do not undergo verb movement. The suggestion to be made in this article is that these ‘‘immobile'' verbs have to fulfill both the requirements imposed on complex verbs of the V° type (=verbs with non-separable prefixes) and the requirements...... are immobile, - why such verbs are not found in Germanic VO-languages such as English and Scandinavian.......Certain complex verbs in Dutch, German, and Swiss German do not undergo verb movement. The suggestion to be made in this article is that these ‘‘immobile'' verbs have to fulfill both the requirements imposed on complex verbs of the V° type (=verbs with non-separable prefixes) and the requirements...

  13. Vero细胞在不同微载体固定化培养中的生长和代谢%Growth and Metabolism of Vero Cells Immobilized Cultured on Various Microcarriers

    Institute of Scientific and Technical Information of China (English)

    刘红; 叶玲玲; 李世崇; 王启伟; 刘兴茂; 陈昭烈

    2012-01-01

    Objective: To investigate the growth and metabolism of Vero cells immobilized cultured on various microcarriers. Methods: Cell viability and cell morphology of the Vero cells cultured in 1% (v/v) newborn bovine serum DMEM/F12, the growth of the Vero cells cultured on 2D MicroHex, Biosilon, Cytodex 1 and Cytopore 1 was evaluated on the viable cell density; The specific consumption rate of glucose (qglc), the specific production rate of lactate (qlac), the specific consumption rate of glutamine (qgln), and the specific consumption rate of glutamate (qglu) were used as the evaluation indexes, the metabolism of the Vero cells cultured on various microcarriers was determined. Results: After 7 d in culture the viable cell density of the Vero cells cultured on 2D MicroHex, Biosilon, Cytodex 1 and Cytopore 1 was 18.4x 105 cells/ml, 21.9×105 cells/ml, 23.9× 105 cells/ml and 16.2× 105 cells/ml, respectively. And, the Vero cells growth on Cytodex 1 formed compact cell layers with distinct cell morphology; Metabolic indexes of the Vero cells cultured on various microcarriers were basically the same. Conclusion: Among these microcarriers, Cytodex 1 is comparatively good for the growth of Vero cells, and could be used as the favor choice of microcarrier for the large-scale cultivation of Vero cells for viral vaccine production.%目的:比较Vero细胞在不同的商品化微载体中固定化培养的生长和代谢.方法:以Vero细胞在含1%新生牛血清的DMEM/F12中培养的细胞形态、活细胞密度和细胞活力为指标,考察Vero细胞在2D MicroHex、Biosilon、Cytodex 1和Cytopore 1微载体固定化培养的细胞生长;以葡萄糖比消耗速率(qglc)、乳酸比生产速率(qlac)、谷氨酰胺比消耗速率(qgln)和谷氨酸比生产速率(qglu)为指标,考察Vero细胞在不同微载体固定化培养的细胞代谢.结果:Vero细胞在2D MicroHex、Biosilon、Cytodex 1和Cytopore 1微载体固定化培养7d的活细胞密度分别为18.4×105

  14. Free-Flow Open-Chamber Electrophoresis

    Science.gov (United States)

    Sharnez, Rizwan; Sammons, David W.

    1994-01-01

    Free-flow open-chamber electrophoresis variant of free-flow electrophoresis performed in chamber with open ends and in which velocity of electro-osmotic flow adjusted equal to and opposite mean electrophoretic velocity of sample. Particles having electrophoretic mobilities greater than mean mobility of sample particles move toward cathode, those with mobilities less move toward anode. Technique applied to separation of components of mixtures of biologically important substances. Sensitivity enhanced by use of tapered chamber.

  15. Covalent co-immobilization of heparin/laminin complex that with different concentration ratio on titanium surface for selectively direction of platelets and vascular cells behavior

    Science.gov (United States)

    Wang, Jian; Chen, Yuan; Liu, Tao; Wang, Xue; Liu, Yang; Wang, Yuan; Chen, Junying; Huang, Nan

    2014-10-01

    Surface biofunctional modification of coronary artery stent to improve the hemocompatibility and selectively accelerate endothelium regeneration but prevent restenosis have been become a new hotspot. For this, a novel method was developed in this work by co-immobilization of Ln and heparin complex on poly-L-lysine modified Ti surface. Take the advantage of the specific interaction between Ln and heparin, Ln and heparin complexes with different concentration ratios were set up for creating different exposure density of these two types of biomolecules. According to biocompatibility evaluation results, the Hep/Ln complexes modified surface displayed less platelet adhesion and activation. Especially, on L(150)H and L(200)H surface, the AT III binding quantity, APTT value and anti-coagulation property of modified surface were significantly promoted. Furthermore, the adherent density and proliferation activity of ECs and EPCs were positively correlated with Ln concentration. Notably, the proliferation of both ECs and EPCs on L(100)H, L(150)H and L(200)H surface were greatly promoted. Another hand, the proliferation activity of SMCs was significantly inhibited on Hep/Ln modified surfaces, which was considered mainly due to the inhibitory effect of heparin to SMCs. According to the existing results, this study demonstrated that in a certain range of heparin and laminin concentration ratio, the biological behavior of platelets, ECs, EPCs and SMCs could be selectively directed. We suggested that this article provided a potential method to construct an adequate platform on a stent surface for accelerate endothelialization with low side effects.

  16. Usage of Capillary Electrophoresis for screening common Hemoglobinopathies

    Directory of Open Access Journals (Sweden)

    2016-06-01

    Full Text Available Hemoglobinopathies are most common inherited disorders in the world approximately 7 percent of the worldwide population and 5-6 percent of population of Iran are carriers. For control of this inherited hemoglobin disorders need to accurate screening by more advanced and more accurate methods. This study explains features of current Iran hemoglobin disorders, nominates the accessible methods for screening them and introduces the capillary zone electrophoresis as a rapid & more accurate method. The required data were extracted of various articles and then for good explanation, current Iran hemoglobinopathies properties were showed in the tables and electropherograms of important hemoglobin disorders in Iran population were provided for help to interpretation results of blood tests by capillary zone electrophoresis method. Hemoglobin disorders are including thalassemias & hemoglobin variants Disruption in the production and malfunction of globin chains cause types of hemoglobin disorders. We cannot introduce one of clinical laboratory tests as critical and basic method for screening and distinguishing types of inherited hemoglobin disorders as alone. For distinguishing the types of them must be prepared enough information and data of the hemoglobin disorders and for more accurate analysis must be used simultaneously different methods as Gel electrophoresis, High performance liquid chromatography, Isoelectric focusing, Capillary zone electrophoresis or molecular tests. The capillary electrophoresis is an accurate and rapid method for screening types of the hemoglobin disorders. Other side this method cannot analyze all of them, so must be used biochemical, biophysical and molecular methods for confirmation the results. This review showed we can use the capillary electrophoresis and HPLC as two complementary methods for hemoglobinopathies screening. We can analyze by the methods more hemoglobin disorders and decrease more laboratory errors. Moreover

  17. Sperm-immobilizing monoclonal antibody to human seminal plasma antigens.

    Science.gov (United States)

    Shigeta, M; Watanabe, T; Maruyama, S; Koyama, K; Isojima, S

    1980-01-01

    Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6783353

  18. High performance enzyme fuel cells using a genetically expressed FAD-dependent glucose dehydrogenase α-subunit of Burkholderia cepacia immobilized in a carbon nanotube electrode for low glucose conditions.

    Science.gov (United States)

    Fapyane, Deby; Lee, Soo-Jin; Kang, Seo-Hee; Lim, Du-Hyun; Cho, Kwon-Koo; Nam, Tae-hyun; Ahn, Jae-Pyoung; Ahn, Jou-Hyeon; Kim, Seon-Won; Chang, In Seop

    2013-06-28

    FAD-dependent glucose dehydrogenase (FAD-GDH) of Burkholderia cepacia was successfully expressed in Escherichia coli and subsequently purified in order to use it as an anode catalyst for enzyme fuel cells. The purified enzyme has a low Km value (high affinity) towards glucose, which is 463.8 μM, up to 2-fold exponential range lower compared to glucose oxidase. The heterogeneous electron transfer coefficient (Ks) of FAD-GDH-menadione on a glassy carbon electrode was 10.73 s(-1), which is 3-fold higher than that of GOX-menadione, 3.68 s(-1). FAD-GDH was able to maintain its native glucose affinity during immobilization in the carbon nanotube and operation of enzyme fuel cells. FAD-GDH-menadione showed 3-fold higher power density, 799.4 ± 51.44 μW cm(-2), than the GOX-menadione system, 308.03 ± 17.93 μW cm(-2), under low glucose concentration, 5 mM, which is the concentration in normal physiological fluid.

  19. MUCOADHESIVE GEL WITH IMMOBILIZED LYSOZYME: PREPARATION AND PROPERTIES

    Directory of Open Access Journals (Sweden)

    Dekina S. S.

    2015-08-01

    Full Text Available The study of non-covalent immobilized lysozyme, as well as physico-chemical and biochemical properties of obtained mucoadhesive gel was the aim of the research. Lysozyme activity was determined by bacteriolytic method (Micrococcus lysodeikticus cells acetone powder was a substrate. Lysozyme immobilization was conducted by the method of entrapment in gel. Enzyme carrier interaction was studied by viscometric, spectrophotometric and spectrofluorimetric methods. Mucoadhesive gel with immobilized lysozyme, possessing antiinflammatory and antimicrobial activities, was prepared. Due to immobilization, protein-polymer complex with the original enzymatic activity was formed. The product is characterized by high mucoadhesive properties, quantitative retaining of protein and bacteriolytic activity, prolonged release of the enzyme, improved biochemical characteristics (extended pH-activity profile, stability in acidic medium and during storage for 2 years, and it is perspective for further studies. The proposed method for lysozyme immobilization in the carboxymethyl cellulose sodium salt gel allows to obtain a stable, highly efficient product, with high adhesive properties for attachment to the mucous membranes, that is promising for use in biomedicine.

  20. Electrostatic protein immobilization using charged polyacrylamide gels and cationic detergent microfluidic Western blotting.

    Science.gov (United States)

    Kim, Dohyun; Karns, Kelly; Tia, Samuel Q; He, Mei; Herr, Amy E

    2012-03-06

    We report a novel protein immobilization matrix for fully integrated microfluidic Western blotting (WB). The electrostatic immobilization gel (EIG) enables immobilization of all proteins sized using cetyl trimethylammonium bromide polyacrylamide gel electrophoresis (CTAB-PAGE), for subsequent electrophoretic probing with detection affinity reagents (e.g., labeled antibodies). The "pan-analyte" capture strategy introduced here uses polyacrylamide gel grafted with concentrated point charges (zwitterionic macromolecules), in contrast to existing microfluidic WB strategies that rely on a sandwich immunoassay format for analyte immobilization and detection. Sandwich approaches limit analyte immobilization to capture of only a priori known targets. A charge interaction mechanism study supports the hypothesis that electrostatic interaction plays a major role in analyte immobilization on the EIG. We note that protein capture efficiency depends on both the concentration of copolymerized charges and ionic strength of the gel buffer. We demonstrate pan-analyte immobilization of sized CTAB-laden model proteins (protein G, ovalbumin, bovine serum albumin, β-galactosidase, lactoferrin) on the EIG with initial capture efficiencies ranging from 21 to 100%. Target proteins fixed on the EIG (protein G, lactoferrin) are detected using antibody probes with signal-to-noise ratios of 34 to 275. The approach advances protein immunoblotting performance through 200× reduction on sample consumption, 12× reduction in assay duration, and automated assay operation, compared to slab-gel WB. Using the microfluidic WB assay, assessment of lactoferrin in human tear fluid is demonstrated with a goal of advancing toward nonbiopsy-based diagnosis of Sjögren's Syndrome, an autoimmune disease.

  1. Ferrous ion oxidation by Thiobacillus ferrooxidans immobilized on activated carbon

    Institute of Scientific and Technical Information of China (English)

    ZHOU Ji-kui; QIN Wen-qing; NIU Yin-jian; LI Hua-xia

    2006-01-01

    The immobilization of Thiobacillus ferrooxidans on the activated carbon particles as support matrix was investigated. Cycling batch operation results in the complete oxidation of ferrous iron in 8 d when the modified 9 K medium is set to flow through the mini-bioreactor at a rate of 0.104 L/h at 25 ℃. The oxidation rate of ferrous iron with immobilized T. ferrooxidans is 9.38 g/(L·h). The results show that the immobilization of T. ferrooxidans on activated carbon can improve the rate of oxidation of ferrous iron. The SEM images show that a build-up of cells of T. ferrooxidans and iron precipitates is formed on the surface of activated carbon particles.

  2. Gelatin Functionalization of Biomaterial Surfaces: Strategies for Immobilization and Visualization

    Directory of Open Access Journals (Sweden)

    Peter Dubruel

    2011-01-01

    Full Text Available In the present work, the immobilization of gelatin as biopolymer on two types of implantable biomaterials, polyimide and titanium, was compared. Both materials are known for their biocompatibility while lacking cell-interactive behavior. For both materials, a pre-functionalization step was required to enable gelatin immobilization. For the polyimide foils, a reactive succinimidyl ester was introduced first on the surface, followed by covalent grafting of gelatin. For the titanium material, methacrylate groups were first introduced on the Ti surface through a silanization reaction. The applied functionalities enabled the subsequent immobilization of methacrylamide modified gelatin. Both surface modified materials were characterized in depth using atomic force microscopy, static contact angle measurements, confocal fluorescence microscopy, attenuated total reflection infrared spectroscopy and X-ray photo-electron spectroscopy. The results indicated that the strategies elaborated for both material classes are suitable to apply stable gelatin coatings. Interestingly, depending on the material class studied, not all surface analysis techniques are applicable.

  3. Immobilized surfactant-nanotube complexes support selectin-mediated capture of viable circulating tumor cells in the absence of capture antibodies.

    Science.gov (United States)

    Mitchell, Michael J; Castellanos, Carlos A; King, Michael R

    2015-10-01

    The metastatic spread of tumor cells from the primary site to anatomically distant organs leads to a poor patient prognosis. Increasing evidence has linked adhesive interactions between circulating tumor cells (CTCs) and endothelial cells to metastatic dissemination. Microscale biomimetic flow devices hold promise as a diagnostic tool to isolate CTCs and develop metastatic therapies, utilizing E-selectin (ES) to trigger the initial rolling adhesion of tumor cells under flow. To trigger firm adhesion and capture under flow, such devices also typically require antibodies against biomarkers thought to be expressed on CTCs. This approach is challenged by the fact that CTCs are now known to exhibit heterogeneous expression of conventional biomarkers. Here, we describe surfactant-nanotube complexes to enhance ES-mediated capture and isolation of tumor cells without the use of capture antibodies. While the majority of tumor cells exhibited weaker rolling adhesion on halloysite nanotubes (HNT) coated with ES, HNT functionalization with the sodium dodecanoate (NaL) surfactant induced a switch to firm cellular adhesion under flow. Conversely, surfactant-nanotube complexes significantly reduced the number of primary human leukocytes captured via ES-mediated adhesion under flow. The switch in tumor cell adhesion was exploited to capture and isolate tumor cells in the absence of EpCAM antibodies, commonly utilized as the gold standard for CTC isolation. Additionally, HNT-NaL complexes were shown to capture tumor cells with low to negligible EpCAM expression, that are not efficiently captured using conventional approaches.

  4. Papain immobilized polyurethane as an ureteral stent material.

    Science.gov (United States)

    Maria Manohar, Cynthya; Doble, Mukesh

    2016-05-01

    Long term use of polyurethane-based ureteral stent is hampered by the development of infection due to the formation of bacterial biofilm and salt deposition. Here papain, is covalently immobilized to polyurethane using glutarldehyde and is investigated as a possible anti-infective ureteral stent material. Fourier transform infrared spectrum confirmed its immobilization. Immobilized enzyme retained 85% of the activity of the free enzyme and about 12% loss of enzyme was observed from the polymer surface in one month. The modified polyurethane showed 8 log reduction in Staphylococcus aureus and 7 log reduction in Escherichia coli live colonies and 3-4 times decrease in the protein and carbohydrate in the biofilms than bare polymer. The amount of calcium and magnesium salts deposited on the polymer surface reduced by 40% after enzyme immobilization. 80% of L6 myoblast cells were viable on this material which indicated that it was noncytotoxic. A linear regression equation with hydrophilicity of the polymer surface and the cell surface hydrophobicity as the two independent variables was able to predict the number of live cells attached on the modified PU. This study indicated the possibility of using such an approach to overcome the problems of ureteral stent associated biofilm and salt encrustation.

  5. Studies on the surface coat of Paramecium aurelia. II. Relationship to the immobilization antigen.

    Science.gov (United States)

    Wyroba, E

    1977-07-11

    Correlations between the presence of surface coat and immobilization antigen of Paramecium tetraurelia were studied. Supravital, partial removal of the surface coat resulted in accelerated response of monobacterially and axenically grown cells to the homologous antiserum. Ciliates pretreated with trypsin or pronase (0.5 mg/ml for 45 min at 0-4 degrees C) were immobilized approximately twice as fast as untreated control cells. The probable localization of at least part, of the immobilization antigen within the surface coat of P. tetraurelia is discussed.