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  • 1
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Company
    Nature biotechnology 5 (1987), S. 800-804 
    ISSN: 1546-1696
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: [Auszug] For some years, there has been great interest in the exploitation of plant cell cultures to produce fine chemicals. With a few exceptions, progress in commercialization has been slow, largely due to the low and/or unstable productivity of many undifferentiated cultures. Recent developments leading ...
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  • 2
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The toxicity of Cinchona alkaloids to cell cultures of C. ledgeriana has been studied in relation to alkaloid uptake and possibilities for selecting high-yielding cell lines. The most toxic, quinine, was completely toxic at 5.5 mM. Both quinine and quinidine were more toxic than their unmethoxylated precursors, cinchonidine and cinchonine. The permanently-charged metho-chlorides of quinine and cinchonidine were less toxic than the parent alkaloids, despite showing similar accumulation ratios in 5-day uptake experiments at sub-toxic concentrations (ca 1.7mM). The toxicity of the natural quinoline alkaloids appears to be a non-specific effect which may be caused by intracellular alkalinisation following uptake of the uncharged bases. The use of precursors of quinine and quinidine as toxic agents for the selection of cell lines with enhanced quinine and quinidine production is ruled out by the lower toxicity of these precursors and by the correlation of an apparently non-specific toxicity with uptake.
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  • 3
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Crown gall initiation on Mentha × piperita var. citrata (Ehrh.) Briq. (mint) was investigated using a range of wild type and mutant strains of Agrobacterium tumefaciens. Axenic transformed shoot cultures of Mentha ‘citrata’ were established on plant stems inoculated with the nopaline strain T37 of Agrobacterium tumefaciens. The presence of T-DNA in the transformed tissues and the absence of bacterial contamination was established by Southern Blot hybridisation, using 32P labelled fragments of the T-DNA and virulence region of the Ti plasmid as probes. The shoot cultures synthesised a mint oil fraction which contained the major terpenes characteristic of the parent plant in quantities similar to those found in intact tissue. Oil glands were observed to be present on the leaves of the transformed culture using scanning electron microscopy.
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  • 4
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The treatment of root cultures of Datura stramonium with copper and cadmium salts at external concentrations of approximately 1mM has been found to induce the rapid accumulation of high levels of sesquiterpenoid defensive compounds, notably lubimin and 3-hydroxylubimin. These compounds were undetectable in unelicited cultures. No net change was seen in the alkaloid content of the system following treatment with Cu2+ or Cd2+, the tropane alkaloid titre apparently being insensitive to elicitation. However, a considerable rapid and, in some instances, reversible release of alkaloid was observed. This resulted in the appearance of up to 50–75% of the total alkaloid in the medium after 40–60 h. Subsequently, in cultures treated with Cu2+ ions, though not in cultures treated with Cd2+ ions, this alkaloid was re-absorbed. These observations show how, in a single system, different groups of secondary products can show distinct differences in their responses to potential elicitors.
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  • 5
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Transformed root lines of Nicotiana species, containing NPTII and Gus genes, were used to study the parameters affecting the use of the Polymerase Chain Reaction as a routine analytical tool for quickly analysing plant transformants for the presence of a foreign gene. The basic reaction mix as described by Cetus Corporation (Saiki 1989) was close to optimal for successful PCR amplification of internal sequences of both NPTII and Gus from genomic plant DNA. The temperature of primer annealing in the PCR protocols was found to be the most important variable, as low temperatures caused amplification of artefact bands and smearing after analysis on ethidium bromide agarose gels. Various formulae for calculating the Tm for binding of primers of various lengths (20–30 bases) are described in relation to predicting suitable annealing temperatures in the PCR. For tobacco species the PCR reaction worked efficiently with up to 2 μg of genomic DNA. However, with DNA from Mentha species (mint), an inhibitor of the PCR process was co-extracted with the DNA which prevented amplification of target sequences, if more than 10 ng of genomic DNA was present in the reaction.
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  • 6
    ISSN: 1432-2048
    Keywords: Datura (root, alkaloid) ; Root culture (alkaloid production) ; Scopolamine synthesis ; Tropane alkaloid synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Using in combination an analysis of (i) the levels of enzyme activities present, (ii) the pool sizes of metabolic intermediates and end products and (iii) the effects of feeding metabolic intermediates, the limitations ℴ flux into tropane alkaloids in a Datura root culture have been examined. This culture, produced by transforming a Datura candida × D. aurea hybrid with Agrobacterium rhizogenes, is found to be highly competent in the biosynthesis of both hyoscyamine and scopolamine as well as a wide range of other hygrine-derived alkaloids. It has been found that, of six enzymes which are involved in this pathway, the two initial activities, ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19), are present at potentially flux-limiting levels, in contrast to those other enzymes assayed which act further down the pathway. An additional limitation to flux, involving the supply of activated acids for condensation with tropine to form the identified tropoyl and tigloyl derivatives, is also indicated from the observed effect of feeding free acids. The relative contribution to flux limitation caused by these two interacting phenomena is inferred from an analysis of the changing relative levels of metabolic intermediates and end products as cultures mature.
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  • 7
    ISSN: 1432-2048
    Keywords: Alkaloid biosynthesis ; Datura ; Putrescine ; N-methyltransferase ; Root (alkaloid biosynthesis) ; Root culture (transformed) ; Tropan alkaloids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transformed root cultures of Datura stramonium, competent in tropane-alkaloid biosynthesis, have been treated with exogenous plant growth regulators. It was found that combinations of α-naphthalene-acetic acid, kinetin (N6-furfurylaminopurine) and 2,4-dichlorophenoxyacetic acid induced de-differentiation, causing both the rooty phenotype and the hyoscyamine-biosynthetic capacity to be lost. Alkaloid biosynthesis disappeared rapidly and prior to the loss of morphological integrity. It was observed that the enzymes ornithine decarboxylase (EC 4.1.1.17), arginine decarboxylase (EC 4.1.1.19) and N-methylputrescine oxidase did not show the increase in level normally associated with subculturing the roots. The level of putrescine N-methyltransferase (EC 2.1.1.53) activity, the first enzyme fully committed to hyoscyamine biosynthesis, rapidly declined, about 80% being lost from the roots within 12h. This activity, although showing some temporary restoration, declined further after a few days, and was totally absent from fully dispersed cultures. N-Methylputrescine oxidase persisted at a low level. Following sub-culture of established de-differentiated lines to plant-growth-regulator-free medium, limited root regeneration occurred. The roots formed showed renewed competence in alkaloid biosynthesis and putrescine N-methyltransferase and N-methylputrescine oxidase activities were restored to their normal levels. The relationship between the morphological state and alkaloid-biosynthetic capacity of the cultures is discussed in relation to the overall control of alkaloid biosynthesis.
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  • 8
    ISSN: 1432-2048
    Keywords: Arginine decarboxylase ; Datura (root, alkaloid) ; Root culture (alkaloid production) ; Hyoscyamine synthesis ; Tropane alkaloid biosynthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The factors by which the endogenous regulation of tropane-alkaloid biosynthesis may be effected have been examined in a transformed root culture of Datura stramonium. Pools of intermediates showed a subculture-related maximal accumulation, as did the enzyme activities by which they are synthesised and/or metabolised. The end-products, principally hyoscyamine and apohyoscyamine, in contrast, accumulated steadily in growing cultures. Feeding putrescine, agmatine or tropine did not enhance alkaloid accumulation, but rather may even have resulted in a lowering of hyoscyamine levels. Similarly, feeding precursors for the tropate moiety of hyoscyamine either had no influence or had a detrimental effect on hyoscyamine accumulation. Under some feeding conditions, intermediates in the pathway from N-methylputrescine up to and including tropine accumulated up to 40-fold. Little effect on early intermediates was found, however, when tropinone or tropine were fed. The expression of the enzyme arginine decarboxylase (EC 4.1.1.19) was particularly sensitive to feed-back repression, both by its product agmatine and by more distant pathway intermediates, notably putrescine and tropine. Some diminution of the levels of putrescine N-methyltransferase (EC 2.1.1.53) and N-methylputrescine oxidase, the first committed enzymes of alkaloid biosynthesis, was also seen with tropine, although only at rather high levels. It is concluded that the pathway is not regulated in a simple manner and that (i) the early enzymes of the pathway are at near rate-limiting levels, (ii) there is a major limitation to flux at the level of the esterification of tropine, and (iii) the level of free tropine may be important in determining the flux into and through the tropane pathway.
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  • 9
    ISSN: 1432-2048
    Keywords: Datura ; Cadaverine ; Putrescine-N-methyltransferase ; Root culture (Agrobacterium-transformed) ; Tropane alkaloid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Putrescine-N-methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes. The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave K m values for putrescine and S-adenosyl-l-methionine of 0.31 mM and 0.10 mM, respectively, and K i values for S-adenosyl-l-homocysteine and N-methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino-trans-but-2-ene. Little activity was observed with 1,4-diamino-cis-but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a K i of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.
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  • 10
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Pre-existing methods for measuring cell or organelle volume based on the selective permeability of biological membranes have been modified to make them suitable for determining the intracellular volume of immoblised cells. When a freely permeable substance (e.g. tritiated water) and an impermeable substance (14C labelled mannitol is often suitable) are mixed with an immobilised cell culture, the two substances are diluted to different degrees. The extent of the difference allows the total intracellular volume of intact cells to be calculated. This volume is shown to be a useful parameter for assessing cell growth. The application of the method to follow membrane integrity and cell viability is also discussed.
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