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characterization of an indian isolate of cucumber mosaic virus infecting egyptian henbane (hyoscyamus muticus l.).a cucumber mosaic virus isolate was found to be associated with mottle crinkle and severe mosaic disease of egyptian henbane (hyoscyamus muticus l.). the virus has been characterized as an indian isolate of cucumber mosaic virus (cmv) based on non-persistent transmission by aphid, presence of 28-nm isometric particles, capsid protein of 26 k and single-stranded tripartite rna genome with a subgenomic rna (rna 4). there was no evidence of satellite rna genome. the isolate showed a strong serologi ...200011155354
expression of vitreoscilla hemoglobin enhances growth of hyoscyamus muticus hairy root cultures.the vitreoscilla hemoglobin gene (vhb) was introduced into hyoscyamus muticus with the aim of investigating its effect on growth and alkaloid production of agrobacterium rhizogenes-induced hairy root cultures. we were able to generate several vhb-expressing hairy root lines with different integration patterns. substantial somaclonal variation was observed in growth and hyoscyamine production amongst both vhb-expressing lines and controls. despite this variation, the growth properties of single l ...200515678373
identifying functional domains within terpene cyclases using a domain-swapping strategy.cyclic terpenes and terpenoids are found throughout nature. they comprise an especially important class of compounds from plants that mediate plant- environment interactions, and they serve as pharmaceutical agents with antimicrobial and anti-tumor activities. molecular comparisons of several terpene cyclases, the key enzymes responsible for the multistep cyclization of c10, c15, and c20 allylic diphosphate substrates, have revealed a striking level of sequence similarity and conservation of exo ...19968692906
somaclonal variation in transformed roots and protoplast-derived hairy root clones of hyoscyamus muticus.substantial somaclonal variation in growth rate, morphology, and alkaloid production of hyoscyamus muticus l. hairy root clones obtained by transformation with four agrobacterium strains was shown. the hyoscyamine content of the root clones (n = 100) obtained from the same origin varied from 0.03 to 0.59% of dry weight. the clones produced 25-320 times less scopolamine than hyoscyamine. the best producing root clone was used as a starting material for protoplast isolation. the hyoscyamine conten ...199817253215
quantitative exploration of the catalytic landscape separating divergent plant sesquiterpene synthases.throughout molecular evolution, organisms create assorted chemicals in response to varying ecological niches. catalytic landscapes underlie metabolic evolution, wherein mutational steps alter the biosynthetic properties of enzymes. here we report the first systematic quantitative characterization of the catalytic landscape underlying the evolution of sesquiterpene chemical diversity. on the basis of our previous discovery of a set of nine naturally occurring amino acid substitutions that functio ...200818776889
biotransformation of hyoscyamine into scopolamine in transgenic tobacco cell cultures.hyoscyamine-6beta-hydroxylase (h6h) catalyses the conversion of hyoscyamine into its epoxide scopolamine, a compound with a higher added value in the pharmaceutical market than hyoscyamine. we report the establishment of tobacco cell cultures carrying the hyoscyamus muticus h6h gene under the control of the promoter camv 35s. the cell cultures were derived from hairy roots obtained via genetically modified agrobacterium rhizogenes carrying the pri and plal21 plasmids. the cultures were fed with ...200716904229
influence of agrobacterium rhizogenes strains on biomass and alkaloid productivity in hairy root lines of hyoscyamus muticus and h. albus *.using leaf explants of in vitro grown hyoscyamus albus and h. muticus plantlets, hairy roots were induced following inoculation with agrobacterium rhizogenes strains a (4) and lba-9402. the transformed roots, appearing after 14 - 17 days incubation on hormone-free ms medium containing 1 g/l cephalexin, were excised and maintained in the same medium. ten randomly selected hairy root lines from each bacterial treatment of the two plant systems were compared for growth and alkaloid production in ha ...199917260237
direct agrobacterium tumefaciens-mediated transformation of hyoscyamus muticus hairy roots using green fluorescent protein.hyoscyamus muticus hairy root segments were infected with agrobacterium tumefaciens ase containing the binary vector pcgn1548 with a green fluorescent protein (gfp) reporter gene under the control of the camv 35s promoter. the roots were incubated on callus-inducing medium to generate transformed cells. transformants were selected on medium containing 50 and 100 mg/l kanamycin and screened by visual inspection for gfp expression. highly fluorescent cells were incubated on phytohormone-free mediu ...199910194405
effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of datura metel and hyoscyamus muticus.in order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the t-dna of the ri plasmid together with the tobacco pmt gene under the control of camv 35s promoter, into the genome of datura metel and hyoscyamus muticus. this gene codes for putrescine:sam n-methyltransferase (pmt; ec. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. hairy root cultures overexpres ...200312493848
simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of hyoscyamus muticus by micellar electrokinetic chromatography.hyoscyamus muticus hairy root clones were established following infection with agrobacterium rhizogenes strains a4, lba-9402 and 15834 and with a. tumefaciens strain c58c1prtgus104. the accumulation of tropane alkaloids hyoscyamine, littorine and scopolamine was evaluated by micellar electrokinetic capillary electrophoresis. littorine was reported for the first time in these clones as well as in the roots of the intact plant and confirmed by collision induced dissociation-mass spectrometry. trop ...200010939356
enhanced secretion of tropane alkaloids in nicotiana tabacum hairy roots expressing heterologous hyoscyamine-6beta-hydroxylase.hyoscyamine-6beta-hydroxylase (h6h; ec 1.14.11.11) catalyses oxidative reactions in the biosynthetic pathway leading from hyoscyamine to the more pharmaceutically valuable tropane alkaloid scopolamine. the h6h gene encoding h6h from hyoscyamus niger was introduced, under the control of the camv 35s promoter, into non-hyoscyamine-producing nicotiana tabacum and hyoscyamine-producing hyoscyamus muticus. the transformation was performed using a binary vector system based on agrobacterium rhizogenes ...200516105856
heterologous expression of vitreoscilla hemoglobin (vhb) and cultivation conditions affect the alkaloid profile of hyoscyamus muticus hairy roots.fast-growing hairy root cultures of hyoscyamus muticus induced by agrobacterium rhizogenes offer a potential production system for tropane alkaloids. oxygen deficiency has been shown to limit growth and biomass accumulation of hairy roots, whereas little experimental data is available on the effect of oxygen on alkaloid production. we have investigated the effect of vitreoscilla hemoglobin (vhb) expression and cultivation conditions on the complete alkaloid profile of h. muticus hairy roots in s ...200616599546
enhanced recovery of solavetivone from agrobacterium transformed root cultures of hyoscyamus muticus using integrated product extraction.the integrated recovery of solavetivone from fungus elicited "hairy root" cultures of hyoscyamus muticus is examined using volatile organic solvents and solid-phase adsorbents in an external loop extraction configuration. hexane and pentane are shown to be toxic when added directly to the culture; however, growth of roots is not inhibited when cultures are exposed to media saturated with these hydrocarbons. solid-phase neutral adsorbents, xad-7 and xad-16, display higher capacity and better sola ...199318613055
a soluble auxin-binding protein from hyoscyamus muticus is a glutathione s-transferase.we have used the photoaffinity label azido-[3h]iaa (5-n3-[7-3h]indole-3-acetic acid), a biologically active analog of indole-3-acetic acid, to identify auxin-binding proteins (abps) in the soluble fraction of hyoscyamus muticus. a 25-kd polypeptide previously described (h. macdonald, a. m. jones, p. j. king [1991] j biol chem 266: 7393-7399) has now been purified to homogeneity by conventional methods. binding of azido-[3h]iaa to the purified protein was reduced by active auxins but not by inact ...19938108497
mycoflora associated with hyoscyamus muticus growing under an extremely arid desert environment (aswan region, egypt).hyoscyamus muticus l. (egyptian henbane) is one of the desert medicinal plants of family solanaceae. the plant produces pharmaceutically important compounds (tropane alkaloids) as secondary metabolites. in the present study, we describe mycoflora of h. muticus grown in four different locations in egyptian southern desert (aswan region): aswan university campus, wadi allaqi down stream part, aswan airport road, and sahari city. eighty-one species and two varieties belonging to 31 genera were isol ...200818383230
reconstitution of plant nitrate reductase by escherichia coli extracts and the molecular cloning of the chla gene of escherichia coli k12.extracts from escherichia coli, wild type and chlb, chlc, chld, chle, and chlg, but not chla mutants, were able to reconstitute the nitrate reductase activity in nicotiana tabacum cnx68 and hyoscyamus muticus ma-2 mutant extracts. because cnx68 and ma-2 lack the molybdenum cofactor required for nitrate reductase activity, these results indicate that the functional chla gene is essential to produce the molybdenum cofactor in e. coli. a clone containing a gene capable of complementing the chla mut ...19836363588
characterization of fluid-flow resistance in root cultures with a convective flow tubular bioreactoragrobacterium transformed root cultures of hyoscyamus muticus were grown in a recirculating 2 l tubular bioreactor system. performance of this convective flow reactor (cfr) was compared to a bubble column (bc) reactor of the same geometry: replicated cfr experiments produced an average tissue concentration of 556 +/- 4 grams fresh weight per liter in 30 d whereas the bubble column produced only 328 +/- 5 grams per liter corresponding to 25.3 +/- 0.0 and 14.3 +/- 0.5 grams dry weight per liter, r ...199810099442
pre-steady-state study of recombinant sesquiterpene cyclases.an escherichia coli expression system was used to generate hexahistidyl-tagged plant sesquiterpene cyclases, which were readily purified by a single affinity chromatographic step. genes for hyoscyamus muticus vetispiradiene synthase (hvs), a chimeric 5-epi-aristolochene synthase (ch3), and a chimeric sesquiterpene cyclase possessing multifunctional epi-aristolochene and vetispiradiene activity (ch4) were expressed in bacterial cells, which resulted in the sesquiterpene cyclases accumulating to 5 ...19979204881
growth of plant root cultures in liquid- and gas-dispersed reactor environments.the growth of agrobacterium transformed "hairy root" cultures of hyoscyamus muticus was examined in various liquid- and gas-dispersed bioreactor configurations. reactor runs were replicated to provide statistical comparisons of nutrient availability on culture performance. accumulated tissue mass in submerged air-sparged reactors was 31% of gyratory shake-flask controls. experiments demonstrate that poor performance of sparged reactors is not due to bubble shear damage, carbon dioxide stripping, ...19937763701
cloning and bacterial expression of a sesquiterpene cyclase from hyoscyamus muticus and its molecular comparison to related terpene cyclases.genomic and cdna clones for vetispiradiene synthase, a sesquiterpene cyclase found in hyoscyamus muticus, were isolated using a combination of reverse transcription-polymerase chain reactions and conventional cloning procedures. rna blot hybridization demonstrated an induction of mrna consistent with the induction of cyclase enzyme activity in elicitor-treated cells, dna blot hybridization indicated a gene family of 6 to 8 members, and bacterial expression of 3 cdna clones indicated that each co ...19957706281
rhizosphere fungi of five plants in egypt.75 species which belong to 25 genera were collected during this investigation. all of these fungi were previously isolated from egyptian soils and other sources. the total count and the number of genera and species in the rhizosphere soil of the 5 plants were regularly higher than in non-rhizosphere soil. the composition of the rhizosphere fungus flora was considerably influenced by the type and the age of the plant. the most prevalent fungi in the rhizosphere of the test plants after 45, 90, an ...19807424047
growth and the uptake of sucrose and mineral ions by transformed root cultures of datura stramonium, datura candida x aurea, datura wrightii, hyoscyamus muticus and atropa belladonna.growth and the uptake of sucrose, anions (cl(-), no3(-), po4(3-), and so4(2-)), and cations (k(+), na(+), mg(2+), ca(2+), and nh4(+)) by transformed roots of datura stramonium, datura wrightii. datura candida x datura aurea hybrid, hyoscyamus muticus, and atropa belladonna were examined during batch culture over 28 days in modified 14 litre stirred tank reactors containing gamborg's b5 salts medium. all cultures completely removed nh4(+) and po4(3-) from the medium, and mg(2+) was totally remove ...199517238087
agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species.two novel techniques improve division and colony formation from protoplasts: 1) plating in agarose stimulates colony formation of protoplasts from a wide range of species. protoplasts from nicotiana tabacum developed to colonies from lower initial population densities in agarose than in agar or liquid. protoplasts from hyoscyamus muticus which do not divide in agar divided and formed colonies in agarose at higher efficiencies than in liquid medium. 2) culture of gel embedded protoplasts in large ...198324258119
over-expression of ice2 stabilizes cytochrome p450 reductase in saccharomyces cerevisiae and pichia pastoris.membrane-anchored cytochrome p450 enzymes (cyps) are a versatile and interesting class of enzymes for industrial applications, as they are capable of regio- and stereoselectively hydroxylating hydrophobic molecules. however, cyp activity requires sufficient levels of suitable cytochrome p450 reductases (cprs) for regeneration of catalytic capacity, which is a bottleneck in many industrial applications. searching for positive effectors of membrane-anchored cyp/cpr function, we transformed and scr ...201525641738
virulence of different agrobacterium strains on hairy root formation of hyoscyamus muticus.hyoscyamus muticus accession was evaluated for its response to inoculation with agrobacterium rhizogenes strains lba9402, a4, 15834, and agrobacterium tumefaciens strain c58 ci prt gus104. the agrobacterium strain used for the transformation has a significant influence on the phenotype of the clone as well as on the growth rate and hyoscyamine production of these root culture clones. the most virulent strains were c58 ci prt gus104 and lba9402. more roots were obtained on lso medium than on b50 ...199524190302
intrinsic oxygen use kinetics of transformed plant root culture.root meristem oxygen uptake, root tip extension rate, and specific growth rate are assessed as a function of dissolved oxygen level for three transformed root cultures. the influence of hydrodynamic boundary layer was considered for all measurements to permit correlation of oxygen-dependent kinetics with the concentration of oxygen at the surface of the root meristem. oxygen uptake rate is shown to be saturated at ambient conditions, and a saturation level of approximately 300 micromole o2/(cm(3 ...201311386869
an unusual root tip formation in hairy root culture of hyoscyamus muticus.hairy root cultures of hyoscyamus muticus were established using agrobacterium rhizogenes atcc 15834. in one out of 8 clones established, an unusual root tip formation was observed after transfer of cultures from half-strength murashige and skoog (1962) to white's medium (1939). this phenomenon was associated with the production of a fine brownish cell suspension culture. hairy root development resumed after transfer of the root tips from white to half-strength murashige and skoog medium. after ...199424193835
building terpene production platforms in yeast.plants and microbes commonly make terpenes and terpenoids in small amounts and as complex mixtures, and their chemical synthesis is often costly and inefficient. hence, there are many efforts to create robust and efficient biological production platforms for this interesting class of molecules. in this study, our effort was directed towards building a yeast production platform using an unbiased genetic selection approach. yeast strain by4741 was subjected to ems mutagenesis, followed by selectio ...201525788404
production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of pichia pastoris.the sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or nootka cypress tree. extraction of (+)-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. in our study, the yeast ...201424747046
exploring the metabolic stability of engineered hairy roots after 16 years maintenance.plants remain a major source of new drugs, leads and fine chemicals. cell cultures deriving from plants offer a fascinating tool to study plant metabolic pathways and offer large scale production systems for valuable compounds - commercial examples include compounds such as paclitaxel. the major constraint with undifferentiated cell cultures is that they are generally considered to be genetically unstable and cultured cells tend to produce low yields of secondary metabolites especially over time ...201627746806
responses of fungi to tropane alkaloids produced by a medicinal plant hyoscyamus muticus (egyptian henbane).antifungal activity of hyoscyamine (hcy) and scopolamine (sco) were determined by tlc-bioautography against fungi associated with h. muticus grown in egypt, and those isolated from other plants grown in japan. all 40 fungal strains were tolerant to sco and sensitive to hcy, exhibiting a growth inhibition zone around the hcy spot on the bioautography plate. the strains were grouped into three types based on the appearance of the inhibition zone: (i) 17 strains exhibiting a clear inhibition zone, ...200919649737
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