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Plant Cell Cultures (plant + cell_culture)
Selected AbstractsAlterations in Taxol Production in Plant Cell Culture via Manipulation of the Phenylalanine Ammonia Lyase PathwayBIOTECHNOLOGY PROGRESS, Issue 6 2002Michelle C. Brincat One approach to increasing secondary metabolite production in plant cell culture is to manipulate metabolic pathways to utilize more resources toward production of one desired compound or class of compounds, such as diverting carbon flux from competing secondary pathways. Since phenylalanine provides both the phenylisoserine side chain and the benzoyl moiety at C-2 of Taxol, we speculated that blockage of the phenylpropanoid pathway might divert phenylalanine into Taxol biosynthesis. We used specific enzyme inhibitors to target the first enzyme in the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL), the critical control point for conversion of l -phenylalanine to trans -cinnamic acid. Cinnamic acid acted quickly in reducing PAL activity by 40,50%, without affecting total protein levels, but it generally inhibited the taxane pathway, reducing Taxol by 90% of control levels. Of the taxanes produced, 13-acetyl-9-dihydro-baccatin III and 9-dihydrobaccatin III doubled as a percentage of total taxanes in C93AD and CO93P cells treated with 0.20 and 0.25 mM cinnamic acid, when all other taxanes were lowered. The PAL inhibitor ,-aminooxyacetic acid (AOA) almost entirely shut down Taxol production at both 0.5 and 1.5 mM, whereas l -,-aminooxy-,-phenylpropionic acid (AOPP) had the opposite effect, slightly enhancing Taxol production at 1 ,M but having no effect at 10 ,M. The discrepancy in the effectiveness of AOA and AOPP and the lack of effect with addition of phenylalanine or benzoic acid derivatives further indicates that the impact of cinnamic acid on Taxol is related not to its effect on PAL but rather to a specific effect on the taxane pathway. On the basis of these results, a less direct route for inhibiting the phenylpropanoid pathway may be required to avoid unwanted side effects and potentially enhance Taxol production. [source] Growth Behavior in Plant Cell Cultures Based on Emissions Detected by a Multisensor ArrayBIOTECHNOLOGY PROGRESS, Issue 4 2004Palle Komaraiah The use of a multisensor array based on chemical gas sensors to monitor plant cell cultures is described. The multisensor array, also referred to as an electronic nose, consisted of 19 different metal oxide semiconductor sensors and one carbon dioxide sensor. The device was used to continuously monitor the off-gas from two plant cell suspension cultures, Morinda citrifolia and Nicotiana tabacum, cultivated under batch conditions. By analyzing the multiarray responses using two pattern recognition methods, principal component analysis and artificial neural networks, it was possible to monitor the course of the cultivations and, in turn, to predict (1) the biomass concentration in both systems and (2) the formation of the secondary metabolite, antraquinone, by M. citrifolia. The results identify the multisensor array method as a potentially useful analytical tool for monitoring plant process variables that are otherwise difficult to analyze on-line. [source] Bioreactor Production of Human ,1 -Antitrypsin Using Metabolically Regulated Plant Cell CulturesBIOTECHNOLOGY PROGRESS, Issue 3 2002Melody M. Trexler Transgenic rice cell cultures, capable of producing recombinant human ,1 -antitrypsin (rAAT), were scaled up from shake flasks to a 5-L bioreactor. The maximum specific growth rates (,max) observed from two bioreactor runs were 0.40 day,1 (doubling time of 1.7 days) and 0.47 day,1 (doubling time of 1.5 days), and the maximum specific oxygen uptake rates were 0.78 and 0.84 mmol O2/(g dw h). Using a metabolically regulated rice ,-amylase (RAmy3D) promoter, signal peptide, and terminator, sugar deprivation turned on rAAT expression, and rAAT was secreted into the culture medium. After 1 day of culture in sugar-free medium, there was still continued biomass growth, oxygen consumption, and viability. Extracellular concentrations of 51 and 40 mg active rAAT/L were reached 1.7 and 2.5 days, respectively, after induction in a sugar-free medium. Volumetric productivities for two batch cultures were 7.3 and 4.6 mg rAAT/(L day), and specific productivities were 3.2 and 1.6 mg rAAT/(g dw day). Several different molecular weight bands of immunoreactive rAAT were observed on immunoblots. [source] ChemInform Abstract: Synthesis of Styrenes Through the Biocatalytic Decarboxylation of trans-Cinnamic Acids by Plant Cell Cultures.CHEMINFORM, Issue 46 2001Masumi Takemoto Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full-size antibody in BY-2 suspension culture,BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010T. Holland Abstract Plant cell cultures have been used as expression hosts for recombinant proteins for over two decades. The quality of plant cell culture-produced proteins such as full-size monoclonal antibodies has been shown to be excellent in terms of protein folding and binding activity, but the productivity and yield fell short of what was achieved using mammalian cell culture, in which the key to gram-per-liter expression levels was strain selection and medium/process optimization. We carried out an extensive media analysis and optimization for the production of the full-size human anti-HIV antibody 2G12 in N. tabacum cv. BY-2. Nitrogen source and availability was found to be one key factor for the volumetric productivity of plant cell cultures. Increased amounts of nitrate in the culture medium had a dramatic impact on protein yields, resulting in a 10,20-fold increase in product accumulation through a combination of enhanced secretion and higher stability. The results were scalable from shake flasks to stirred-tank bioreactors, where the maximum yield per cultivation volume was 8,mg,L,1 over 7 days. During the stationary phase, antibody levels were 150-fold higher in nitrogen-enriched medium compared to standard medium. The enhanced medium appeared not to affect antibody quality and activity, as determined by Western blots, surface plasmon resonance binding assays and N -glycan analysis. Biotechnol. Bioeng. 2010;107: 278,289. © 2010 Wiley Periodicals, Inc. [source] Bioreactor strategies for improving production yield and functionality of a recombinant human protein in transgenic tobacco cell culturesBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Ting-Kuo Huang Abstract Plant cell culture production of recombinant products offers a number of advantages over traditional eukaryotic expression systems, particularly if the product can be targeted to and purified from the cell culture broth. However, one of the main obstacles is product degradation by proteases that are produced during cell culture, and/or the loss of biological activity of secreted (extracellular) products as a result of alteration in the protein conformation. Because proteolysis activity and target protein stability can be significantly influenced by culture conditions, it is important to evaluate bioprocess conditions that minimize these effects. In this study, a bioreactor strategy using a protocol involving pH adjustment and medium exchange during plant cell culture is proposed for improving the production of functional recombinant ,1 -antitrypsin (rAAT), a human blood protein, produced using several alternative expression systems, including a Cauliflower mosaic virus (CaMV) 35S constitutive promoter expression system, a chemically inducible, estrogen receptor-based promoter (XVE) expression system, and a novel Cucumber mosaic virus (CMV) inducible viral amplicon (CMViva) expression system developed by our group. We have demonstrated that higher medium pH help reduce protease activity derived from cell cultures and improve the inherent stability of human AAT protein as well. This strategy resulted in a fourfold increase in the productivity of extracellular functional rAAT (100 µg/L) and a twofold increase in the ratio of functional rAAT to total rAAT (48%) in transgenic N. benthamiana cell cultures using a chemically inducible viral amplicon expression system. Biotechnol. Bioeng. 2009;102: 508,520. © 2008 Wiley Periodicals, Inc. [source] Alterations in Taxol Production in Plant Cell Culture via Manipulation of the Phenylalanine Ammonia Lyase PathwayBIOTECHNOLOGY PROGRESS, Issue 6 2002Michelle C. Brincat One approach to increasing secondary metabolite production in plant cell culture is to manipulate metabolic pathways to utilize more resources toward production of one desired compound or class of compounds, such as diverting carbon flux from competing secondary pathways. Since phenylalanine provides both the phenylisoserine side chain and the benzoyl moiety at C-2 of Taxol, we speculated that blockage of the phenylpropanoid pathway might divert phenylalanine into Taxol biosynthesis. We used specific enzyme inhibitors to target the first enzyme in the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL), the critical control point for conversion of l -phenylalanine to trans -cinnamic acid. Cinnamic acid acted quickly in reducing PAL activity by 40,50%, without affecting total protein levels, but it generally inhibited the taxane pathway, reducing Taxol by 90% of control levels. Of the taxanes produced, 13-acetyl-9-dihydro-baccatin III and 9-dihydrobaccatin III doubled as a percentage of total taxanes in C93AD and CO93P cells treated with 0.20 and 0.25 mM cinnamic acid, when all other taxanes were lowered. The PAL inhibitor ,-aminooxyacetic acid (AOA) almost entirely shut down Taxol production at both 0.5 and 1.5 mM, whereas l -,-aminooxy-,-phenylpropionic acid (AOPP) had the opposite effect, slightly enhancing Taxol production at 1 ,M but having no effect at 10 ,M. The discrepancy in the effectiveness of AOA and AOPP and the lack of effect with addition of phenylalanine or benzoic acid derivatives further indicates that the impact of cinnamic acid on Taxol is related not to its effect on PAL but rather to a specific effect on the taxane pathway. On the basis of these results, a less direct route for inhibiting the phenylpropanoid pathway may be required to avoid unwanted side effects and potentially enhance Taxol production. [source] Amino acid profiling in plant cell cultures: An inter-laboratory comparison of CE-MS and GC-MSELECTROPHORESIS, Issue 9 2007Brad J. Williams Abstract A CE-MS method for metabolic profiling of amino acids was developed and used in an integrated functional genomics project to study the response of Medicago truncatula liquid suspension cell cultures to stress. This project required the analysis of more than 500 root cell culture extracts. The CE-MS method profiled 20 biologically important amino acids. The CE-MS method required no sample derivatization prior to injection and used minimal sample preparation. The method is described in terms of CE and MS operational parameters, reproducibility of migration times and response ratios, sample preparation, sample throughput, and reliability. This method was then compared with a previously published report that used GC-MS metabolic profiling for the same tissues. The data reveal a high level of similarity between the CE-MS and GC-MS amino acid profiling methods, thus supporting these as complementary technologies for metabolomics. We conclude that CE-MS is a valid alternative to GC-MS for targeted profiling of metabolites, such as amino acids, and possesses some significant advantages over GC-MS. [source] Metabolism of fluoranthene in different plant cell cultures and intact plantsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 5 2000Marit Kolb Abstract The metabolism of fluoranthene was investigated in 11 cell cultures of different plant species using a [14C]-labeled standard. Most species metabolized less than 5% of fluoranthene to soluble metabolites and formed less than 5% nonextractable residues during the standardized 48-h test procedure. Higher metabolic rates were observed in lettuce (Lactuca sativa, 6%), wheat (Tricitum aestivum, 9%), and tomato (Lycopersicon esculentum, 15%). A special high metabolic rate of nearly 50% was determined for the rose species Paul's Scarlet. Chromatographic analysis of metabolites extracted from aseptically grown tomato plants proved that the metabolites detected in the cell cultures were also formed in the intact plants. Metabolites produced in tomato and rose cells from [14C]-fluoranthene were conjugated with glucose, glucuronic acid, and other cell components. After acid hydrolyses, the main metabolite of both species was 1-hydroxyfluoranthene as identified by gas chromatography,mass spectrometry and high-performance liquid chromatography with diode array detection. The second metabolite formed by both species was 8-hydroxy-fluoranthene. A third metabolite in tomatoes was 3-hydroxyfluoranthene. [source] Techniques for oxygen transfer measurement in bioreactors: a reviewJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 8 2009S Suresh Abstract Oxygen is the most essential requirement for aerobic bioprocesses. The microbial growth in a bioreactor depends upon the oxygen transfer rate (OTR). The OTR is widely used to study the growth behavior of microbial and plant cell cultures. The mass transfer coefficient (kLa) determines the magnitude of the OTR. There are many techniques for measuring oxygen concentration and OTR in bioreactors. Zirconia, electrochemical, infrared, ultrasonic and laser cells are used to measure oxygen concentration in the liquid medium. Optical sensors are better alternatives to measure oxygen concentration in small bioreactors. Sulfite oxidation and gassing-out methods with a Clark-type electrode have been used for OTR measurements in bioreactors. Many new novel techniques have evolved recently for intermittent and continuous online measurement of OTR/kLa in various types of bioreactors. The present paper gives an overview of various measurement techniques and their limitations and/or suitability for measurement of OTR/kLa in various kinds of bioreactors, especially small bioreactors. Copyright © 2009 Society of Chemical Industry [source] Uptake of nicotine from suspension culture of Nicotiana tabacum by molecularly imprinted polymersJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2010Mohamed Salaheldin A. Abdelkader Abstract Objectives The aim was to use molecularly imprinted polymers (MIPs) for the selective recovery of nicotine in plant cell cultures. MIPs can selectively uptake nicotine from suspension cultures of N. tabacum, and therefore may be useful for improving levels of secondary metabolites in plant cell cultures. Methods Suspension cultures of N. tabacum were initiated from callus and maintained in liquid Murashige and Skoog (MS) media containing 3% w/v sucrose, 0.1 mg/l ,-naphthaleneacetic acid acid (NAA) and 0.25 mg/l kinetin. Tween 80 at 1% was used for permeabilisation of cell cultures. Pre-weighed XAD-2 and two types of synthesized polymers, MIPs (A and B with one and two functional monomers, respectively) and corresponding non-imprinted polymers (NIPs), A and B, were introduced aseptically into the permeabilised suspension cultures of N. tabacum, the nicotine contents of polymers were determined by gas chromatography and the adsorption yield of polymers were determined. Key findings Cell cultures of N. tabacum accumulated nicotine alkaloid intracellularly in varying levels, 6.8,14.9 mg/l fresh weight. MIPs were able to uptake 50,70% of released nicotine in suspension cultures of N. tabacum, whereas XAD-2 recovered only 30,40%. The total levels of accumulated nicotine were enhanced up to 20 mg/l by simultaneous use of Tween 80 and MIPs. Conclusions The findings indicate the potential use of MIPs to uptake nicotine from suspension cultures of N. tabacum, and increase productivity of secondary metabolites in plant cell cultures. [source] Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full-size antibody in BY-2 suspension culture,BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010T. Holland Abstract Plant cell cultures have been used as expression hosts for recombinant proteins for over two decades. The quality of plant cell culture-produced proteins such as full-size monoclonal antibodies has been shown to be excellent in terms of protein folding and binding activity, but the productivity and yield fell short of what was achieved using mammalian cell culture, in which the key to gram-per-liter expression levels was strain selection and medium/process optimization. We carried out an extensive media analysis and optimization for the production of the full-size human anti-HIV antibody 2G12 in N. tabacum cv. BY-2. Nitrogen source and availability was found to be one key factor for the volumetric productivity of plant cell cultures. Increased amounts of nitrate in the culture medium had a dramatic impact on protein yields, resulting in a 10,20-fold increase in product accumulation through a combination of enhanced secretion and higher stability. The results were scalable from shake flasks to stirred-tank bioreactors, where the maximum yield per cultivation volume was 8,mg,L,1 over 7 days. During the stationary phase, antibody levels were 150-fold higher in nitrogen-enriched medium compared to standard medium. The enhanced medium appeared not to affect antibody quality and activity, as determined by Western blots, surface plasmon resonance binding assays and N -glycan analysis. Biotechnol. Bioeng. 2010;107: 278,289. © 2010 Wiley Periodicals, Inc. [source] Effect of the plant peptide regulator, phytosulfokine-,, on the growth and Taxol production from Taxus sp. suspension culturesBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Beum Jun Kim Abstract Phytosulfokine-, (PSK-,) is a small plant peptide (5 amino acids) that displays characteristics typically associated with animal peptide hormones. PSK-, was originally isolated based on its mitogenic activity with plant cultures; it has been reported to increase production of tropane alkaloids from Atropa belladonna, although its general influence on secondary metabolite production is unknown. The studies reported in this article were initiated to evaluate the effects of PSK-, supplementation on production of TaxolÔ (paclitaxel) from plant cell cultures of Taxus sp. particularly when methyl jasmonate (MeJA) is added as an elicitor of secondary metabolism. The response to PSK-, supplementation was cell line dependent. Taxus cuspidata P93AF showed no statistically significant response to PSK-, supplementation while Taxus canadensis C93AD and T. cuspidata PO93X displayed a concentration-dependent response (up to 100 nM PSK-, added in first 24 h of culture) with a decrease in initial growth rate, an increase in cell density (dry weight/fresh weight), and increased Taxol production. More remarkably with T. canadensis (C93AD), a very strong synergistic response of PSK-, (100 nM) and methyl jasmonate (MeJA, 100 µM) elicitation was observed, resulting in Taxol level of 35.3,±,2.1 mg/L or 1.83,±,0.02 mg Taxol/g dry cell weight achieved at day 21, a level of approximately 10-fold higher than for either treatment by itself. Although the level of Taxol production achieved is not remarkable, this synergistic treatment was able to partially revive taxane production in cultures that have lost productivity due to extended time (over 10 years) in continuous subculture. © 2006 Wiley Periodicals, Inc. [source] Growth Behavior in Plant Cell Cultures Based on Emissions Detected by a Multisensor ArrayBIOTECHNOLOGY PROGRESS, Issue 4 2004Palle Komaraiah The use of a multisensor array based on chemical gas sensors to monitor plant cell cultures is described. The multisensor array, also referred to as an electronic nose, consisted of 19 different metal oxide semiconductor sensors and one carbon dioxide sensor. The device was used to continuously monitor the off-gas from two plant cell suspension cultures, Morinda citrifolia and Nicotiana tabacum, cultivated under batch conditions. By analyzing the multiarray responses using two pattern recognition methods, principal component analysis and artificial neural networks, it was possible to monitor the course of the cultivations and, in turn, to predict (1) the biomass concentration in both systems and (2) the formation of the secondary metabolite, antraquinone, by M. citrifolia. The results identify the multisensor array method as a potentially useful analytical tool for monitoring plant process variables that are otherwise difficult to analyze on-line. [source] Comparative Metabolism of ,- and ,- Peptides in the Insect Heliothis virescens and in Plant Cells of Black Mexican Sweet MaizeCHEMISTRY & BIODIVERSITY, Issue 9 2004Rob Lind The tripeptide H-Val-Ala-Leu-OH and the N -Ac-tetrapeptide amide Ac-Thr-Lys-Trp-Phe-NH2, and their , -peptidic counterparts H- ,3hVal- ,3hAla- ,3hLeu-OH and Ac- ,3hThr-(S),2hLys- ,3hTrp- ,3hPhe-NH2, respectively, have been injected into Heliothis virescens larvae and added to cell cultures of black mexican sweet maize. The body liquids of the larvae and the supernatant of the plant cell cultures were sampled 0, 2, 3, 6, 17, and/or 24,h after application and analyzed by LC/MS. While the two , -peptides were degraded rapidly in these environments, the concentration of the , -peptides was found to decrease very slowly. Thus, ca. 60% of the original amount of the , -tetrapeptide was detected in the liquids of the insect after 24,h. The plant cells did not seem to make use of the , -peptides at all, whereas, the , -tripeptide completely disappeared from the supernatant after 3,h. Thus, we have demonstrated, for the first time, the high stability of , -peptides against degradation and metabolism in an insect and a plant. Especially remarkable is the persistence of the , -tetrapeptide with its functionalised and, thus, ,metabolisable' side chains of Thr, Lys, Trp, and Phe in the insect larvae, which are known to have a high level of activity of oxidizing enzymes. The results described here match those of ADME investigations with radioactively labeled , -peptides in rats, where essentially complete stability has been observed, while environmental microorganisms have been found to biodegrade , -peptides, albeit slowly. Possible implications of these findings for biomedical and pest-control applications are proposed. [source] | |||||||||||||