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permeabilization of plant cells: (31)p nmr studies on the permeability of the tonoplast.a suspension culture of catharanthus roseus has been used to study the permeability of cell membranes after treatment with various concentrations of a permeabilizing agent (dmso). the uptake and release (after permeabilization) of inorganic phosphate (pi) by cells have been investigated by (32)p radiotracer and non-invasive phosphorus-31 nmr experiments. these studies have demonstrated that measurements of the pi-efflux from plant cells provide a reliable measure of the permeability of the tonop ...198624247956
alkaloid production in root and shoot cultures of catharanthus roseus.root and shoot cultures were induced from seedlings of catharanthus roseus. root cultures and cell suspension cultures derived from the root cultures showed similar alkaloid spectra to the roots of intact plants. the alkaloid spectra of shoot cultures and the aerial parts of intact plants were similar to each other but different from those of root and suspension cultures. illumination affected the alkaloid spectra of shoot cultures. shoot cultures produced 3',4'anhydrovinblastine, an immediate p ...198717269072
increased accumulation of indole alkaloids by some cell lines of catharanthus roseus in response to addition of vanadyl sulphate.vanadyl sulphate (10-500 mg/l), when added to cell suspension cultures of catharanthus roseus stimulated increased intracellular accumulation of catharanthine and ajmalicine. this response was demonstrated in both flask and fermenter (30 litre) systems. the response varied, and depended upon cell line, concentration of vanadyl sulphate and the stage of the growth phase at which the cells were treated. this process has the potential to increase the yield and reduce the production time for commerc ...198724248497
characterization of a novel n-methyltransferase (nmt) from catharanthus roseus plants : detection of nmt and other enzymes of the indole alkaloid biosynthetic pathway in different cell suspension culture systems.young leaves from catharanthus roseus plants contain a novel n-methyltransferase which transfers the methyl group from s-adenosyl-l-methionine specifically to position 1 of (2r, 3r)-2,3-dihydro-3-hydroxytabersonine, producing the n-methylated product. the enzyme shows a high degree of specificity toward substrates containing a reduced double bond at position 2,3 of tabersonine derivatives but the more substituted n-desmethyldeacetylvindoline did not act as a substrate. the enzyme catalyses the t ...198724248932
subcellular localization of enzymes involved in indole alkaloid biosynthesis in catharanthus roseus.the subcellular localization of enzymes involved in indole alkaloid biosynthesis in leaves of catharanthus roseus has been investigated. tryptophan decarboxylase and strictosidine synthase which together produce strictosidine, the first indole alkaloid of this pathway, are both cytoplasmic enzymes. s-adenosyl-l-methionine: 16-methoxy-2,3-dihydro-3-hydroxytabersonine-n-methyltransferase which catalyses the third to last step in vindoline biosynthesis could be localized in the chloroplasts of cath ...198716665811
immunoanalytical methods for screening vindoline from catharanthus roseus cell cultures. 19873444865
alkaloid formation by habituated and tumorous cell suspension cultures of catharanthus roseus.habituated and tumorous catharanthus roseus cells grown in the absence of hormones accumulated indole alkaloids. total alkaloids and alkaloid pattern were the same when cells were cultured in medium without hormones or in alkaloid production medium with and without indole acetic acid. treatment of cells with pythium homogenate as elicitor did not increase total alkaloids or change the pattern of alkaloids produced. when either habituated or tumorous cells were grown in 1b5 medium after gamborg e ...198724248757
stimulation of indole alkaloid production in cell suspension cultures of catharanthus roseus by abscisic acid.when added to suspension cultures of catharanthus roseus, abscisic acid (aba) stimulated intracellular accumulation of the indole alkaloids catharanthine and ajmalicine in both flask and 30 litre fermenter-scale systems. the response varied, and depended upon the cell line, the concentration and source of the aba, and the growth phase at which the cells were treated. precise timing of aba addition to cells in a 301 fermenter resulted in a catharanthine yield of 85.25 mg/l after 10 days of cultiv ...198717269070
ajmalicine, serpentine, and catharanthine accumulation in catharanthus roseus bioreactor cultures.a 141, stirred-tank bioreactor was used to investigate the effect of 7% glucose solutions on catharanthus roseus suspension cultures. measurement of oxygen uptake rate indicated that alkaloid accumulation occurred primarily during a 20-day transition period between growth-oriented metabolism and maintenance-oriented metabolism. exposure of the cells to light during this period stimulated catharanthine accumulation and triggered a switch from ajmalicine accumulation to serpentine accumulation. in ...198717269046
time-course studies in indole alkaloid accumulation and changes in tryptophan decarboxylase and strictosidine synthase activities: a comparison in three strains of catharanthus roseus cells.three different strains of catharanthus roseus cells were compared during one subculture with regard to tryptophan, tryptamine, ajmalicine, serpentine contents and tryptophane decarboxylase (tdc) (4) and strictosidine synthase activities. the strains differed greatly in their accumulation of tryptamine and alkaloid. the tdc of all three strains showed the highest activity during the growth phase and declined sharply at the end of this phase. on the contrary, strictosidine synthase activity was t ...198717269044
[antitumor and toxic effects of amotin].an indole alkaloid isolated from catharanthus roseus in the soviet union and named amotin differs in terpenoid moiety structure of the indole part of the molecule from the foreign preparation vinblastine and its soviet analog rosevine. in experimental study amotin has exhibited a high antileukemic activity which was more expressed than that of vinblastine. tolerant doses of amotin cause moderate reversible morphological alterations in hematopoietic organs, gastrointestinal mucosa, liver, kidney, ...19873691399
increased synthesis of ajmalicine and catharanthine by cell suspension cultures of catharanthus roseus in response to fungal culture-filtrates.the ammonium sulfate-precipitated fraction from mycelia and culture-filtrates and the crude, cell-free culture filtrates from the growth medium of the fungi chrysosporium palmorum, eurotium rubrum, micromucor isabellina, and pythium aphanidermatum when aseptically added to cell suspensions of cantharanthus roseus caused a rapid and dramatic increase in indole alkaloid biosynthesis. up to 400 micrograms/l ajmalicine and 600 micrograms/l catharanthine were detected in c. roseus cell suspension gro ...19873619437
elicitor-mediated induction of tryptophan decarboxylase and strictosidine synthase activities in cell suspension cultures of catharanthus roseus.treatment of one cell line (no. 615) of catharanthus roseus c.v. little delicata with an elicitor preparation of autoclaved and homogenized pythium aphanidermatum culture resulted in rapid accumulation of indole alkaloids. alkaloid formation was preceded by rapid transient increases in the extractable activities of the enzymes tryptophan decarboxylase and strictosidine synthase. the induction of these two enzyme activities occurred when cells were transferred to alkaloid production medium or tre ...19873579315
resistance to pythium aphanidermatum in diploids and induced autotetraploids of catharanthus roseus.resistance to pythium aphanidermatum (which causes die-back and collar and root rot) in seven diploid and four induced autotetraploid lines of catharanthus roseus was studied in the field during 1983 to 1985. the tetraploid lines were, depending on the year of evaluation, about 13 to 155, 18 to 218, and 17 to 366 times more resistant than diploid lines, when resistance was measured in terms of plant mortality, infection rate, and area under disease curve, respectively. on an average over three y ...198817265286
alkaloid production by transformed root cultures of catharanthus roseus.transformed roots of catharanthus roseus were obtained following infection of detached leaves with agrobacterium rhizogenes. roots would not grow in full strength gamborg's b5 medium but would grow satisfactorily if the medium was diluted to one half strength. little alkaloid appeared in the growth medium but root tissue contained a high level and wide variety of alkaloids. ajmalicine, serpentine, vindolinine and catharanthine were prominent components. vinblastine could also be detected by a co ...198824241871
induction of triterpene biosynthesis by elicitors in suspension cultures of tabernaemontana species.treatment of suspension cultures of some tabernaemontana species (apocynaceae) with elicitors (e.g. cellulase, candida albicans) result in a rapid de novo production of antimicrobial active triterpenes. the triterpenes are identified as ursene carboxylic acid derivatives. these triterpenes are not produced by an elicited cell suspension culture of catharanthus roseus, another apocynaceae.198824241415
developmental regulation of enzymes of indole alkaloid biosynthesis in catharanthus roseus.developing seedlings of catharanthus roseus were analyzed for appearance of tryptophan decarboxylase (tdc), strictosidine synthase (ss), n-methyltransferase (nmt) and o-acetyltransferase (dat) enzyme activities. ss enzyme activity appeared early after germination and was present throughout most of the developmental study. tdc activity was highly regulated and peaked over a 48 hour period achieving a maximum by day of 5 of seedling development. both tdc and ss were present in all tissues of the s ...198816665928
formation of vinblastine in multiple shoot culture of catharanthus roseus. 19883375331
auxin induces rapid changes in phosphatidylinositol metabolites.the plant growth-hormone auxin (indole-3-acetic acid, iaa) is involved in regulating such diverse processes as cell elongation, cell division and differentiation. the sequence of events leading to the various phenomena is still poorly understood. both changes in extra- and intracellular ph (refs 1-4) and selective transcription are known to be induced by auxin. evidence for auxin receptors at the plasmalemma membrane has been reported, but the signal transduction pathway is not known, for this n ...19883257546
stimulation of indole alkaloid content in vanadium-treated catharanthus roseus suspension cultures.addition of vanadyl sulphate to cell suspensions of catharanthus roseus was found to increase ajmalicine, catharanthine, and tryptamine levels. up to 500 microg/g dry weight catharanthine and 131.0 microg/g dry weight ajmalicine were detected in vanadyl sulphate-treated cells. this represents an approximate increase of 50% over control levels. this stimulation was found to be dependent upon the concentration of vanadyl sulphate administered and upon the cell age. high tryptamine levels were not ...198817265225
induced net ca(2+) uptake and callose biosynthesis in suspension-cultured plant cells.in suspension-cultured cells of glycine max and catharanthus roseus, marked callose synthesis can be induced by digitonin and chitosan. leakage of a limited pool of electrolytes precedes callose formation, k(+) representing the major cation lost. poly-l-ornithine, as well as the ionophores a 23187 and ionomycin, also induces some callose synthesis but to a lesser extent. digitonin increases the net uptake of ca(2+) from the external buffer with a time course parallel to callose synthesis but lag ...198824226184
diffusion of sucrose and yohimbine in calcium alginate gel beads with or without entrapped plant cells.the diffusion characteristics of sucrose, a nutrient, and yohimbine, a secondary metabolite, in alginate gel beads, with or without entrapped periwinkle (catharanthus roseus) or apple (malus domestica) cells, were investigated. effective diffusivities of both solutes in the gel beads were determined by two different methods from transient concentration changes in well-stirred solutions where the beads were suspended. the linear plot method developed in this work is easy to use and requires no da ...198818587800
bioreactor considerations for secondary metabolite production from plant cell tissue culture: indole alkaloids from catharanthus roseus.batch shake flask studies with catharanthus roseus demonstrated that alkaloid production commenced only after growth had slowed or ceased. to obtain high alkaloid productivities for extended periods, a hormone-free production medium was used. to develop a readily scalable process, both immobilized and suspended cell systems were studied. in the immobilized cell systems, growth, glucose utilization, and alkaloid production were suppressed; for the case of membrane entrapped cells this suppression ...198818584698
characterization of alkaloid uptake by catharanthus roseus (l.) g. don protoplasts.the accumulation of alkaloids by protoplasts of catharanthus roseus (l.) g. don var. little bright eye was studied to determine the specificity of uptake and the role of ion trapping in the storage of alkaloids. accumulation of the indole alkaloids vindoline, ajmalicine, tabersonine, and vinblastine was found to be biphasic, with an initial burst of uptake followed by a slow, prolonged phase of accumulation. the concentration and ph dependence of the initial burst of uptake for vindoline suggest ...198816666154
adhesion of catharanthus roseus cells to surfaces: effect of substrate hydrophobicity. 198818587809
the degrees of polymerization and n-acetylation of chitosan determine its ability to elicit callose formation in suspension cells and protoplasts of catharanthus roseus.partially and fully deacetylated chitosan fragments and oligomers were compared for their potency to elicit formation of the 1.3-β-glucan callose in suspension-cultured cells and protoplasts of catharanthus roseus (line 385). chitosan oligomers induced little callose formation, while callose synthesis increased with the degree of polymerization of chitosan up to several thousand corresponding to a molecular mass near 10(6) da. at a comparable degree of polymerization, partially n-acetylated chit ...198924212905
diffusible factors essential for epidermal cell redifferentiaion in catharanthus roseus.during postgenital tissue fusions, some plant epidermal cells redifferentiate into parenchyma, a different cell type. diffusible factors cause this response in the fusing gynoecium of the madagascar periwinkle (catharanthus roseus). surgical manipulations of the gynoecium showed that epidermal cells from normally nonfusing surfaces could trasmit and respond to the diffusible factors. furthermore, the diffusible fators could be trapped in agar-impregnated barriers, as shown by the redifferentiati ...198917769401
mass spectral evidence of the occurrence of vindoline in heterotrophic cultures of catharanthus roseus cells.vindoline concentrations in the leaves of 70 catharanthus roseus of 3 cultivars were analyzed by hplc, and 3 plants were selected for starting callus cultures on different media. when the initial calli were analyzed using a vindoline-specific ria, the assay suggested a vindoline content of about 10 (-5)% dry weight for one-third of the first 60 cultures examined. due to the unexpectedly high incidence of vindoline-positive calli, the screening programme was discontinued and efforts were concentr ...198917262331
regulation of vacuolar ph of plant cells: i. isolation and properties of vacuoles suitable for p nmr studies.for the first time, the (31)p nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar ph and the inorganic phosphate content. catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and v ...198916666513
carrier-mediated uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid in vacuoles isolated from catharanthus roseus cells.the uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid (macc), the conjugated form of the ethylene precursor, into vacuoles isolated from catharanthus roseus cells has been studied by silicone layer floatation filtering. the transport across the tonoplast of macc is stimulated fourfold by 5 millimolar mgatp, has a k(m) of about 2 millimolar, an optimum ph around 7, and an optimum temperature at 30 degrees c. several effectors known to inhibit atpase (n,n'-dicyclohexylcarbodiimide) and to c ...198916667182
how suspension cultures of catharanthus roseus respond to oxygen limitation: small-scale tests with applications to large-scale cultures.the effect of oxygen supply on the growth of suspension cultures of catharanthus roseus in erlenmeyer flasks was investigated. below a critical oxygen supply rate the culture could not survive. by increasing the oxygen supply, a point is reached where the culture survives but no growth is possible. at higher oxygen supply rates there is a regime where both growth rate and the maximum biomass concentration increase with oxygen supply. eventually there comes a point where no further increase in bi ...198918588199
electrochemical detection of indole alkaloids of catharanthus roseus in high-performance liquid chromatography.hydrodynamic voltammograms for indole and five indole alkaloids with different amino functions were obtained in order to evaluate the applicability of high-performance liquid chromatography (hplc) with coulometric detection to these compounds. with the exception of serpentine, which has a quaternary nitrogen in its structure, all the compounds were oxidised and gave net signals of greater than 25 na pmol(-1) at potentials of between +0.2 and +0.85 v versus a solid pd electrode in an acetate-buff ...19892619069
modification of catharanthus roseus alkaloids: a lactone derived from 17-deacetylvinblastine.a spirolactone with anti-tumor activity is formed during the reaction of paraformaldehyde with 17-deacetylvinblastine. the compound was identified by nmr techniques. the chemotherapeutic activity was assessed.19892813571
regulation of vacuolar ph of plant cells: ii. a p nmr study of the modifications of vacuolar ph in isolated vacuoles induced by proton pumping and cation/h exchanges.the vacuolar ph and the trans-tonoplast deltaph modifications induced by the activity of the two proton pumps h(+)-atpase and h(+)-ppase and by the proton exchanges catalyzed by the na(+)/h(+) and ca(2+)/h(+) antiports at the tonoplast of isolated intact vacuoles prepared from catharanthus roseus cells enriched in inorganic phosphate (y mathieu et al 1988 plant physiol [in press]) were measured using the (31)p nmr technique. the h(+)-atpase induced an intravacuolar acidification as large as 0.8 ...198916666525
phase-specific polypeptides and poly(a) rnas during the cell cycle in synchronous cultures of catharanthus roseus cells.this study shows an overall analysis of gene expression during the cell cycle in synchronous suspension cultures of catharanthus roseus cells. first, the cellular cytoplasmic proteins were fractionated by two-dimensional gel electrophoresis and visualized by staining with silver. seventeen polypeptides showed qualitative or quantitative changes during the cell cycle. second, the rates of synthesis of cytoplasmic proteins were also investigated by autoradiography by labeling cells with [(35)s]met ...198916666641
genetic transformation ofcatharanthus roseus g. don byagrobacterium rhizogenes.catharanthus roseus plantlets were inoculated with differentagrobacterium rhizogenes strains. this plant species is known to produce secondary metabolites and axenic hairy-root cultures are an alternative to extraction of plant tissue for the compounds which are synthesized in roots. hairy root lines were established from inoculations with the agropine strain 15834 and transformed plants were obtained after spontaneous regeneration. phenotypic alterations of both the root system and the aerial p ...198924232985
assay of strictosidine synthase from plant cell cultures by high-performance liquid chromatography.an hplc assay is described for the enzyme strictosidine synthase in which the formation of strictosidine and the decrease of tryptamine can be followed at the same time. in cell cultures of catharanthus roseus significant amounts of strictosidine glucosidase activity were detected. in crude preparations, the strictosidine synthase reaction is therefore best measured by the secologanin-dependent decrease of tryptamine. in this way, the specific synthase activity in a cell free extract was found t ...19892742131
quantitative determination of vinblastine in tissue cultures of catharanthus roseus by radioimmunoassay.a radioimmunoassay has been developed for the quantitative determination of small amounts of vinblastine in tissue cultures of catharanthus roseus (l.) g. don. antibody was obtained by the immunization of rabbits against a conjugate of vinblastine with bovine serum albumin. the antibody had a high affinity (ka = 1.2 x 10 (9)l/mol) and specificity for vinblastine. the usable range of standard curve for assay was 0.5-10 ng/ml. crude alkaloid extracts of tissue cultures could be assayed and many sa ...198917262413
diacylglycerol kinase from suspension cultured plant cells : purification and properties.diacylglycerol kinase (atp:1,2-diacylglycerol 3-phosphotransferase, ec 2.7.1.107) from suspension-cultured catharanthus roseus cells was extracted from a membrane fraction with 0.6% triton x-100 and 150 millimolar nacl and was purified about 900-fold by deae-cellulose, blue sepharose, gel permeation, and phenyl-sepharose chromatography. the enzyme is obviously membrane bound as activity in the cytosol could not be detected. in the presence of detergents such as triton x-100 (3-[3-cholamidopropyl ...198916666963
immunological detection and quantitation of tryptophan decarboxylase in developing catharanthus roseus seedlings.l-tryptophan decarboxylase (tdc) (ec 4.2.1.27) enzyme activity was induced in cell suspension cultures of catharanthus roseus after treatment with a pythium aphanidermatum elicitor preparation. the enzyme was extracted from lyophilized cells containing high levels of tdc and the protein was purified to homogeneity. the pure protein was used to produce highly specific polyclonal antibodies, and an enzyme-linked immunosorbent assay (elisa) was developed to quantitate the level of tdc antigen durin ...198916667047
homogeneous strictosidine synthase isoenzymes from cell suspension cultures of catharanthus roseus.four separable isoforms of strictosidine synthase, which catalyze condensation of tryptamine with secologanin to form strictosidine, were purified to homogeneity from cultured cells of catharanthus roseus and from leaves of c. roseus plants. these enzymes are distinguished by their isoelectric points as well as by their catalytic properties. the specific activity of the main form (isoform iii) is 104 nkat/mg, k (cat) = 3.2 s (-1). the relative molecular mass is 31 kda as estimated by gel filtrat ...198917262473
molecular cloning and analysis of cdna encoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases.the sequence of a cdna clone that includes the complete coding region of tryptophan decarboxylase (ec 4.1.1.28, formerly ec 4.1.1.27) from periwinkle (catharanthus roseus) is reported. the cdna clone (1747 base pairs) was isolated by antibody screening of a cdna expression library produced from poly(a)+ rna found in developing seedlings of c. roseus. the clone hybridized to a 1.8-kilobase mrna from developing seedlings and from young leaves of mature plants. the identity of the clone was confirm ...19892704736
glycaemic effects of traditional european plant treatments for diabetes. studies in normal and streptozotocin diabetic mice.twelve plants used for the traditional treatment of diabetes mellitus in northern europe were studied using normal and streptozotocin diabetic mice to evaluate effects on glucose homeostasis. the plants were administered in the diet (6.25% by weight) and/or as decoctions or infusions in place of drinking water, to coincide with the traditional method of preparation. treatment for 28 days with preparations of burdock (arctium lappa), cashew (anacardium occidentale), dandelion (taraxacum officinal ...19892743711
deduced amino acid sequence of bovine aromatic l-amino acid decarboxylase: homology to other decarboxylases.nucleotide sequence of cdna for bovine aromatic l-amino acid decarboxylase (aadc) was analyzed. the deduced amino acid sequence of bovine aadc shows 57% identity to drosophila aadc and 37% to plant catharanthus roseus aadc. the 7-amino acid sequence of the pyridoxal phosphate binding site is completely conserved among drosophila, pig and bovine aadc. aadc primary structure also shows high homology to that of feline glutamic acid decarboxylase.19902166204
the discovery of the vinca alkaloids--chemotherapeutic agents against cancer.in folklore medicine, extracts of the leaves of the subtropical plant catharanthus roseus (l.) g. don (sometimes known as madagascar periwinkle) were reputed to be useful in the treatment of diabetes. this review describes how attempts to verify the antidiabetic properties of the extracts led instead to the discovery and isolation of two complex indole alkaloids, vinblastine and vincristine, which are used in the clinical treatment of a variety of cancers. the two alkaloids, although structurall ...19902085431
nonradioactive screening method for isolation of disease-specific probes to diagnose plant diseases caused by mycoplasmalike organisms.dna fragments of tomato big bud (bb) mycoplasmalike organism (mlo) in diseased periwinkle plants (catharanthus roseus l.) were cloned to psp6 plasmid vectors and amplified in escherichia coli jm83. a nonradioactive method was developed and used to screen for mlo-specific recombinants. cloned dna probes were prepared by nick translation of the mlo recombinant plasmids by using biotinylated nucleotides. the probes all hybridized with nucleic acid from bb mlo-infected, but not healthy, plants. resu ...199016348195
high levels of tryptamine accumulation in transgenic tobacco expressing tryptophan decarboxylase.a full-length complementary dna clone encoding tryptophan decarboxylase (tdc; ec 4.1.1.28) from catharanthus roseus (de luca v, marineau c, brisson n [1989] proc natl acad sci usa 86: 2582-2586) driven by the camv 35s promoter was introduced into tobacco (nicotiana tabacum) to direct the synthesis of the protoalkaloid tryptamine from endogenous tryptophan. young, fully expanded leaves of camv 35s-tdc transformed plants had from four to 45 times greater tdc activity than did controls. tryptamine ...199016667846
tests for transmission of prunus necrotic ringspot and two nepoviruses by criconemella xenoplax.in two of three trials, detectable color reactions in elisa for prunus necrotic ringspot virus (pnrsv) were observed for criconemella xenoplax handpicked from the root zone of infected peach trees. criconemella xenoplax (500/pot) handpicked from root zones of peach trees infected with pnrsv failed to transmit the virus to cucumber or peach seedlings. the nematode also failed to transmit tomato ringspot (tomrsv) or tobacco ringspot viruses between cucumbers, although xiphinema americanum transmit ...199019287748
a reversible carrier mediates the transport of malate at the tonoplast of catharanthus roseus cells.the conditions of malate transport were defined in tonoplast vesicles purified from a microsomal homogenate of catharanthus roseus cells by preparative free-flow electrophoresis. isolated vesicles exhibited malate transport when the membranes were prepared by grinding the cells in a homogenisation medium only buffered in the acidic ph range. by using vesicles energized artificially by an imposed ph gradient (acid interior), it was shown that malate is actively accumulated in response to the gene ...19902262004
identification of non-equilibrium glycolytic reactions in suspension-cultured plant cells.levels of all enzymes and metabolites involved in glycolysis were determined in suspension-cultured catharanthus roseus cells. from both the maximum catalytic activities of the enzymes and comparisons of mass-action ratios with the apparent equilibrium constants for the reactions, it is concluded that the reactions catalysed by hexokinase, fructokinase, phosphofructokinase and pyruvate kinase are far from equilibrium, whereas the other reactions including that catalysed by pyrophosphate-fructose ...19902223831
isolation and characterization of a 2-oxoglutarate dependent dioxygenase involved in the second-to-last step in vindoline biosynthesis.young leaves from catharanthus roseus plants contain the enzymes which convert the monoterpenoid indole alkaloid, tabersonine by three hydroxylations, two methylations, and one acetylation step to vindoline. a novel direct enzyme assay has been developed for a hydroxylase involved in vindoline biosynthesis, which catalyzes the c4-hydroxylation of 2,3-dihydro-3-hydroxy-n(1)-methyltabersonine to the 3,4-dihydroxy derivative. the enzyme showed an absolute requirement for 2-oxoglutarate and enzymati ...199016667836
nucleotide sequence of a cdna encoding the vacuolar protein strictosidine synthase from catharanthus roseus. 19902395663
effects of long-term preservation on growth and productivity of panax ginseng and catharanthus roseus cell cultures.cell cultures of panax ginseng and catharanthus roseus producing secondary metabolites were preserved in liquid nitrogen or under mineral oil for six months. the growth behaviour and the ability of the cultures to produce ginsenosides or indole alkaloids were measured after a recovery period and compared with cultures maintained by frequent subcultivation during the same period. neither growth kinetics nor the degree of vacuolization during growth were affected by the long term preservation. som ...199024226696
specific differences in tolerance to exogenous berberine among plant cell cultures.the tolerance of plant cells to exogenously administered berberine, an antimicrobial isoquinoline alkaloid, was studied using berberine-producing and nonproducing cell suspension cultures. both coptis japonica and thalictrum flavum cells, which have an intrinsic ability to synthesize berberine, took up exogenous berberine from the culture medium by an energy-requiring active transport to accumulate it exclusively in vacuoles. by contrast, t. minus cells, which excrete indigenous berberine mostly ...199024226596
purification and characterization of acetylcoenzyme a: deacetylvindoline 4-o-acetyltransferase from catharanthus roseus.the enzyme acetylcoenzyme a:deacetylvindoline 4-o-acetyltransferase (ec 2.3.1.-) (dat), which catalyzes the final step in vindoline biosynthesis in catharanthus roseus, was purified 3300-fold using ammonium sulfate precipitation followed by gel filtration, anion exchange, hydroxyapatite, and affinity chromatographies. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) of the purified dat showed the presence of two major proteins having mr values of 33,000 and 21,000, whereas na ...19902350183
gas chromatographic-mass spectrometric analysis of major indole alkaloids of catharanthus roseus. 19902355069
development of a strategy to control the dissolved concentrations of oxygen and carbon dioxide at constant shear in a plant cell bioreactor.to examine the effects of volatile components on plant cell growth, a bioreactor control system was developed to simultaneously control the dissolved concentrations of both oxygen and carbon dioxide. the first step in this work was to develop a mathematical model to account for gas-liquid mass transfer; biological utilization and production of o(2) and co(2); and the series of chemical reactions of co(2) in water. using this model and dynamic measurements for dissolved o(2) and co(2), it was obs ...199018592487
photochemical one-pot synthesis of vinblastine and vincristine.the naturally occurring cytostatic dimer alkaloids vinblastine 1 and vincristine 2 were photochemically synthesized in slightly acidic aqueous solution from the monomer alkaloids catharanthine 3 and vindoline 4. multi-centre reactions should obviously be involved and some of the principal photochemistry-associated phenomena here discussed are quite likely to be characteristic even to the biosynthetic reactions yielding vinblastine 1 and vincristine 2 in the cells of catharanthus roseus.19902367548
monoclonal antibodies to bis-indole alkaloids of catharanthus roseus and their use in enzyme-linked immuno-sorbent-assays.high affinity monoclonal antibodies directed against bis-indole alkaloids from catharanthus roseus were produced. once characterized, they were used to develop sensitive enzyme-linked-immuno-sorbent-assays, enabling us to determine minute quantities of alkaloids related to vinblastine in plant material.19901366691
purification of acetyl-coa: 17-o-deacetylvindoline 17-o-acetyltransferase from catharanthus roseus leaves.the enzyme acetyl-coa: 17-o-deacetylvindoline 17-o-acetyltransferase which terminates vindoline biosynthesis has been isolated from catharanthus roseus leaves, further characterized and purified to homogeneity by three step column chromatography and subsequent preparative isoelectric focusing. kinetic properties concerning the enzyme reaction are discussed. five multiple forms of the acetyl-transferase could be observed, each consisting of two subunits. this enzyme is now the best characterized ...199024232684
optimal temperature control for a structured model of plant cell culture.this article calculates optimal open-loop temperature trajectories that maximize the average rate of product synthesis of a plant cell culture. it uses a previously published five-state mathematical model which describes the growth and product synthesis of a batch plant cell suspension culture of catharanthus roseus under temperature control. the optimal open-loop temperatures maximize the final product concentration for predefined fermentation periods. a single switch in temperature is shown by ...199018592517
alkaloid secondary products from catharanthus roseus cell suspension.plant cell culture, the growth of plant cells on solid medium or in liquid, was originally used to study the physiology and biochemistry of plants without the complication of the whole plant. however, it was soon found that plant cell cultures were often capable of producing compounds characteristic of the original plant. of particular interest were those compounds known as secondary products. secondary products are poorly defined, but in general are products that are not essential for growth, b ...199021390635
identification of indole alkaloids of catharanthus roseus with liquid chromatography/mass spectrometry using collision-induced dissociation with the thermospray ion repeller.fragmentation of protonated molecular ions produced from catharanthine, tabersonine, ajmalicine and serpentine in a high-performance liquid chromatography (hplc) thermospray ion source was studied. collision-induced dissociation (cid) of the compounds was achieved by merely increasing the repeller potential; i.e. no discharge current was applied and filament was not on. proportion of fragments to the protonated molecular ions increased with the potential at the 180-350 v range studied, but overa ...19902400851
characterization of tonoplast-enriched vesicles isolated by gradient density fractionation of suspension-cultured cells.a tonoplast-enriched microsomal fraction was isolated from catharanthus roseus cells. it was characterized by structural and functional criteria. this fraction presented a homogeneous size distribution as shown by quasi-elastic light scattering and a homogeneous density on self-generated gradients of percoll. the mean diameter of the vesicles was estimated to be 0.30 micron and the buoyant density around 1.04 g/ml. sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of its polypep ...19902139317
isolation of catharanthus alkaloids by solid-phase extraction and semipreparative hplc.a combination of moderate-pressure chromatography on c18 sorbent and preparative hplc is developed for rapid isolation of alkaloids from catharanthus roseus. the procedure is optimized for vindoline and catharanthine with respective yields of 3 and 2 mg per 1 g of dried leaves of the plant. the methodology is also applied for identification of the above and other alkaloids from cultured plant cells.19902079546
development of bioreactors for the culture of surface immobilized plant cells.the scaleup of the technique of plant cell surface immobilization was performed successfully in specifically designed laboratory size bioreactors. the immobilizing matrix was formed into a vertically wound spiral providing for a high immobilizing area-to-volume ratio (0.8-1.2 cm(-1)). a modified airlift and a mechanically stirred vessel delivered a best bioreactor performance characterized by low biomass frothing and highly efficient plant cell attachment and retention (>or=96%). the growth of c ...199018592566
production of indole alkaloids by surface immobilized c. roseus cells.a technique was developed to surface immobilize plant cells and was scaled up in laboratory size bioreactors. this technique was shown not to hinder the biosynthetic potential of catharanthus roseus immobilized cells and to induce a partial release (300 microg/l) of serpentine into the culture medium contrary to suspension cultured cells. the release pattern seemed to follow the biosynthesis trends of the product. this release mechanism could be stimulated by a factor of 10 within 2 h by increas ...199018592562
proton/phosphate stoichiometry in uptake of inorganic phosphate by cultured cells of catharanthus roseus (l.) g. don.upon absorption of phosphate, cultured cells of catharanthus roseus (l.) g. don caused a rapid alkalinization of the medium in which they were suspended. the alkalinization continued until the added phosphate was completely exhausted from the medium, at which time the ph of the medium started to drop sharply toward the original ph value. phosphate exposure caused the ph of the medium to increase from ph 3.5 to values as high as 5.8, while the rate of phosphate uptake was constant throughout (10- ...199016667491
effect of thermal power plant emissions on catharanthus roseus l. 19902354262
an investigation into the role of initial k(l)a on the growth and alkaloid accumulation by cultures of catharanthus roseus.the effect of initial k(l)a on the batch culture of catharanthus roseus id1 in a 12.5-l bioreactor has been examined. optimum k(l)a values were found for both growth and alkaloid accumulation. different sparger designs were used to alter the k(l)a values. high k(l)a values caused increased aggregation of the cultures, depressed biomass yields, and altered patterns of alkaloid accumulation.199118597378
isolation of genes that are preferentially expressed at the g(1)/s boundary during the cell cycle in synchronized cultures of catharanthus roseus cells.a cdna library was screened for genes that may be involved in the progression of the cell cycle of cells of higher plants. the catharanthus roseus l. (g) don. cells were synchronized by the double phosphate starvation method, and a lambdagt11 cdna library was prepared using poly(a)(+) rna from cells in the s phase of the cell cycle. two independent sequences, cyc02 and cyc07, were identified by differential screening. the levels of cyc02 and cyc07 mrnas increased dramatically, but transiently, a ...199116667998
effect of culture process on alkaloid production by catharanthus roseus cells. ii. immobilized cultures.two processes for the production of indole alkaloids 2 l surface-immobilized bioreactor cultures of catharanthus roseus cells using zenk's alkaloid production medium (apm) were evaluated. the 1-stage process consisted of inoculating apm containing bioreactors and incubating for 15 d. the 2-stage process involved inoculating growth medium-containing bioreactors, growing the immobilized cultures for a certain period of time and subsequently replacing this medium with apm. the production stage whic ...19911367689
effect of culture process on alkaloid production by catharanthus roseus cells. i. suspension cultures.the processes for production of indole alkaloids in shake flask suspension cultures of catharanthus roseus cells using zenk's alkaloid production medium (apm) were evaluated. the 1-stage process consisted of inoculating apm and incubating for 15 days. the 2-stage process involved 6 d of cultivation in growth medium followed by 15 d of incubation in apm. growth, main nutrient consumption and alkaloid production were monitored. both culture processes produced approximately 20 g dw per 1 biomass. h ...19911367684
effect of near-ultraviolet light on alkaloid production in multiple shoot cultures of catharanthus roseus. 199117226190
expression of enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants.strictosidine, a precursor to over 1000 indole alkaloids including the anti-tumor drugs vinblastine, vincristine, and camptothecin, is produced by the condensation of tryptamine and secologanin. strictosidine synthase, the enzyme responsible for this condensation, is the first committed step in the indole-alkaloid pathway. we have introduced a modified cdna encoding strictosidine synthase from catharanthus roseus (l.) don. (mcknight et al. 1990, nucl. acids res. 18, 4939) driven by the camv 35s ...199124186336
growth and stoichiometry of a catharanthus roseus cell suspension culture grown under nitrogen-limiting conditions.the uptake of carbohydrate and nitrate by catharanthus roseus cell suspension cultures was studied in relation to biomass production in shake flasks. biomass production was similar when using either 6, 12, 18, or 24 mm nitrate as the nitrogen source and 20 g l(-1) sucrose as the carbon source. in all cases, maximum biomass production was reached when carbohydrates were entirely consumer by the cells. apparent biomass yields, y(x/s) and y(x/n) were 0.49 g biomass g(-1) glucose equivalent and 0.23 ...199118604877
polyamines and the cell cycle of catharanthus roseus cells in culture.investigation was made on the effect of partial depletion of polyamines (pas), induced by treatment with inhibitors of the biosynthesis of pas, on the distribution of cells at each phase of the cell cycle in catharanthus roseus (l.) g. don. cells in suspension cultures, using flow cytometry. more cells treated with inhibitors of arginine decarboxylase (adc) and ornithine decarboxylase (odc) were accumulated in the g(1) phase than those in the control, while the treatment with an inhibitor of spe ...199116668290
preparation of sealed tonoplast and plasma-membrane vesicles from catharanthus roseus (l.) g. don. cells by free-flow electrophoresis.highly purified tonoplast and plasmamembrane vesicles were isolated from microsomes of catharanthus roseus (l.) g. don. by preparative free-flow electrophoresis. the relative amounts of tonoplast and plasma-membrane vesicles in the total microsomes varied with the ph of the grinding medium. the most electronegative fractions were identified as tonoplast using nitrate-inhibited, azide-resistant mg(2+)-atpase and pyrophosphatase activities as enzyme markers. the least electronegative fractions wer ...199124194236
large-scale (20 l) culture of surface-immobilized catharanthus roseus cells.surface-immobilized c. roseus cell cultures were grown in a 20-l modified airlift bioreactor operated at 0.51 vvm (kla approximately 8 h-1) under various gassing regimes [air, 2% (v/v) and 5% co2]. extracellular ammonium, phosphate, and nitrate ions as well as carbohydrate uptake and ph value of the medium were monitored together with on-line dissolved oxygen concentration, conductivity of the medium, and carbon dioxide production rate (cpr) of the cultures. cultures supplemented with 2% co2 sho ...19911367999
stimulated indole alkaloid release from catharanthus roseus immobilized cultures. initial studies.vacuolar sequestration of valuable secondary metabolites remains the major limitation to the use of immobilization technology for large scale plant-cell-based bioprocesses, which otherwise may be a more efficient culture system than suspension for this biomass. in this initial study, the release of indole alkaloids produced by immobilized catharanthus roseus cells cultured in zenk's alkaloid production medium was evaluated. unstimulated alkaloid release in immobilized cultures reached levels of ...19911367690
regulation of dna synthesis and cell division by polyamines in catharanthus roseus suspension cultures.various inhibitors of polyamine biosynthesis were used to study the role of polyamines in dna synthesis and cell division in suspension cultures of catharanthus roseus (l.) g. don. arginine decarboxylase (adc; ec 4.1.1.19) was the major enzyme responsible for putrescine production. dl α-difluoromethylarginine inhibited adc activity, cellular putrescine content, dna synthesis, and cell division. the effect was reversible by exogenous putrescine. ornithine decarboxylase (odc; ec 4.1.1.17) activity ...199124221490
secondary metabolite biosynthesis in cultured cells of catharanthus roseus (l.) g. don immobilized by adhesion to glass fibres.suspension-cultured cells of catharanthus roseus (l.) g. don were immobilized on glass fibre mats and cultivated in shake flasks. the highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. the increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways an ...19911367318
induction of catharanthine synthesis and stimulation of major indole alkaloids production by catharanthus roseus cells under non-growth-altering treatment with pythium vexans extracts.a catharanthus roseus cell line was selected that synthesised catharanthine exclusively under elicitation.from the first day of culture, treatment with very low concentrations of a pythium extract did not alter the growth of the suspension but, within 24 hours, induced the synthesis of catharanthine and stimulated the production of ajmalicine. kinetic analysis showed that serpentine then began to accumulate and that all of these effects lasted more than 7 days. elicitation also induced changes i ...199124226159
plant cell growth under different levels of oxygen and carbon dioxide.an experimental system, in which gases of known composition were passed through flasks, was used to systematically study the effects of oxygen and carbon dioxide on plant cell growth. as expected, oxygen limiting conditions resulted in suppressed growth of catharanthus roseus cultures. oxygen limitations did not alter the amount of cell mass produced per gram of sugar consumed which suggests that the production of fermentative metabolites was limited. varying levels of carbon dioxide were observ ...199124226158
first field isolation of wound tumor virus from a plant host: minimal sequence divergence from the type strain isolated from an insect vector.a new strain of wound tumor virus (wtv) has been isolated from a periwinkle plant (catharanthus roseus) that was among several used as bait plants in a blueberry field. the 12 segments of double-stranded rna of the viral genome were isolated directly from infected tissue and found to have mobilities through agarose gels that were identical to those of the type strain wtv. coupled complementary dna (cdna) and polymerase chain reactions (pcr) primed with oligonucleotides complementary to the termi ...19911962460
isolation and characterization of intergeneric somatic hybrids in the apocynaceae family.protoplasts were isolated from callus cultures of rauwolfia serpentina benth., rhazya stricta decaisne, and catharanthus roseus (l.) g. don, or from leaves of vinca minor l. protoplast isolation, culture, and fusion techniques as well as hybrid screening systems were developed for these species, and hybrids were obtained. hybrid combinations were rauwolfia + vinca, rauwolfia + catharanthus, rauwolfia + rhazya, and catharanthus + vinca. for hybrid isolation, the physiological complementation meth ...199124213445
strategies for the genetic manipulation of alkaloid-producing pathways in plants.increasingly, as a result of recent biochemical work, there exists a realistic possibility of taking a molecular genetic approach to the manipulation of alkaloid-producing pathways in plant tissue cultures. in the pathways forming indole alkaloids in catharanthus roseus, tropane alkaloids in datura and hyoscyamus species, and nicotine in nicotiana species, recent studies have identified a number of key enzymes and at least some of the factors that regulate their levels of activity. such knowledg ...199117226220
studies on the optimization of growth and indole alkaloid production by hairy root cultures of catharanthus roseus.catharanthus roseus hairy root cultures, genetically transformed with agrobacterium rhizogenes, produce a wide variety of indole alkaloids. the effect of sucrose, phosphate, nitrate, and ammonia concentrations on growth and indole alkaloid production of c. roseus hairy root cultures were studied by using statistical experimental designs and linear regression analysis. contradictory effects of these nutrients on growth and indole alkaloid production were found. the maximal growth was obtained by ...199118600658
effect of fungal homogenate, enzyme inhibitors and osmotic stress on alkaloid content of catharanthus roseus cell suspension cultures.the addition of aspergillus niger homogenate to catharanthus roseus cell suspension cultures produced an increment of more than 60% in the alkaloid content of two different cell lines. the use of an inhibitor of phenylalanine ammonia lyase, i. e. cinnamic acid, along with the homogenate, resulted in an appearance of 90% of the alkaloids in the medium. furthermore, even in the absence of fungal homogenate, there was a marked increase in the alkaloid content. the exposure of the cells to an osmoti ...199124221325
genetic interrelatedness among clover proliferation mycoplasmalike organisms (mlos) and other mlos investigated by nucleic acid hybridization and restriction fragment length polymorphism analyses.dna was isolated from clover proliferation (cp) mycoplasmalike organism (mlo)-diseased periwinkle plants (catharanthus roseus (l.) g. don.) and cloned into psp6 plasmid vectors. cp mlo-specific recombinant dna clones were biotin labeled and used as probes in dot hybridization and restriction fragment length polymorphism analyses to study the genetic interrelatedness among cp mlo and other mlos, including potato witches'-broom (pwb) mlo. results from dot hybridization analyses indicated that both ...199116348604
molecular cloning of the gene for plant proliferating-cell nuclear antigen and expression of this gene during the cell cycle in synchronized cultures of catharanthus roseus cells.a cdna library was screened for plant proliferating-cell nuclear antigen (pcna) from catharanthus roseus (periwinkle). a lambda gt11 cdna library was constructed using poly(a)-rich rna isolated from the cells in the s phase. a cdna clone for pcna was isolated by using a rice genomic clone, pcj-1, which contains pcna-related gene sequences. the cdna contains an open reading frame of 804 nucleotides, encoding a protein of 268 amino acids with a molecular mass of 29,765 da. when conservative substi ...19911902790
identification of a novel s-phase-specific gene during the cell cycle in synchronous cultures of catharanthus roseus cells.the cell-cycle specific cdnas were isolated from a cdna library prepared from cells in the s phase in the synchronous cultures of catharanthus roseus. one of the isolated genes, which we refer to as cyc07, was analyzed in detail. the full-length cdna of cyc07 contains an open reading frame of 735 nucleotides, encoding a protein of 245 amino acids with a molecular weight of 28,356 da. the protein predicted from the nucleotide sequence is highly basic, as are mammalian histones. cyc07 mrna was det ...19911844881
uptake and accumulation of ajmalicine into isolated vacuoles of cultured cells of catharanthus roseus (l.) g. don. and its conversion into serpentine.isolated vacuoles from ajmalicine-producing cell suspensions of catharanthus roseus accumulated the alkaloid ajmalicine. dissipation of the transtonoplast ph gradient with nigericin abolished ajmalicine accumulation, whereas dissipation of the transtonoplast potential with valinomycin had no effect. addition of mg-atp resulted in a higher ajmalicine accumulation. serpentine produced by the cells was largely recovered in isolated vacuoles; in contrast, ajmalicine was lost. ajmalicine was converte ...199124193617
visualization of vacuoplasts in isolated vacuole preparations from mesophyll protoplasts of periwinkle [catharanthus roseus (l.) g. don].a procedure was developed for the rapid detection of vacuoplasts in vacuole preparations isolated from mesophyll protoplasts of catharanthus roseus (l.) g. don (periwinkle). the procedure relies on the staining of surface carbohydrates on the plasma membrane surrounding vacuoplasts with fluorescein-labeled lectins. when isolated under conditions of constant osmotic strength, approximately 15-20% of the vacuoles isolated showed surface labeling with fitc-agglutinin from abrus precatorius. isolati ...199224203146
coordinated regulation of two indole alkaloid biosynthetic genes from catharanthus roseus by auxin and elicitors.catharanthus roseus (periwinkle) produces a wide range of terpenoid indole alkaloids, including several pharmaceutically important compounds, from the intermediate strictosidine. the complete mrna sequence for the enzyme strictosidine synthase (sss) was determined. comparison of the primary structure of the encoded protein with the amino-terminal sequence of purified sss indicated the presence of a signal peptide of 31 amino acids in the putative primary translation product. sss is encoded by a ...19921600148
auxin rapidly down-regulates transcription of the tryptophan decarboxylase gene from catharanthus roseus.the enzyme tryptophan decarboxylase (tdc) (ec 4.1.1.28) converts tryptophan into tryptamine, and thereby channels primary metabolites into indole alkaloid biosynthesis. the production of these secondary metabolites in suspension cells of catharanthus roseus depends on medium composition. of the possible variables, we investigated the effect of hormones on the expression of the tdc gene in cell cultures. omission of naa from the growth medium resulted in accumulation of tdc mrna. the addition of ...19921600147
sensitive detection and identification of mycoplasma-like organisms in plants by polymerase chain reactions.dna amplification by polymerase chain reactions (pcr) was employed to detect host plant infection by several mycoplasmalike organisms (mlos), including the aster yellows (ay), dwarf aster yellows (day), and periwinkle little leaf (0-1) mlos. for pcr, two pairs of oligonucleotide primers, designated ay18pm and ay19pm, respectively, were synthesized on the basis of partial sequences of cloned ay mlo dna fragments ay18 and ay19. reaction mixtures containing primer pair ay18pm yielded a dna product ...19921510676
the pharmacology of extinction.it is impossible to predict what compounds of pharmacological interest may be present in an unexamined species. the extinction of such species may result, therefore, in the loss of therapeutically significant compounds. the fact that science will never know what has been lost does not lessen the significance of the loss. a number of species are discussed to exemplify the potential loss. ginkgo biloba is an ancient plant, apparently saved from a natural extinction by human intervention. from this ...19921453701
a gene family homologous to the s-phase specific gene in higher plants is essential for cell proliferation in saccharomyces cerevisiae.previously we reported the isolation and characterization of the gene, cyc07, which was specifically expressed in the s phase during the cell cycle in synchronous cell division cultures of the higher plant, catharanthus roseus. we found that the yeast saccharomyces cerevisiae contains two closely related genes which show a high degree of similarity (about 64% at the amino acid level) to cyc07 of c. roseus. site-directed disruption mutations demonstrated that the two yeast genes, homologous to cy ...19921451783
purification of an indole alkaloid biosynthetic enzyme, strictosidine synthase, from a recombinant strain of escherichia coli.the gene for the indole alkaloid biosynthetic enzyme, strictosidine synthase, of catharanthus roseus has been cloned into an inducible escherichia coli expression vector using an expression cassette polymerase chain reaction technique. induction of the gene resulted in overexpression of the enzyme which accumulated mainly as insoluble inclusion bodies. denaturation and refolding of the insoluble protein resulted in the ability to purify up to 6 mg of active enzyme from a single liter of cell cul ...19921330135
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