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PubMed1994

Agglutination of "Streptococcus milleri" by lectins.

The agglutination of 218 clinical isolates and three ATCC type strains of "Streptococcus milleri" was tested with 25 different lectins from plants and fungi. An agglutination reaction with one or more lectins was observed with 42 isolates when the cells were untreated. After trypsinisation of the bacteria, 109 strains yielded a positive reaction and after boiling the bacterial cells at pH2, 218 isolates were agglutinated. As an overall result of our experiments with untreated, trypsinised and boiled cells, 17, 37 and 45 different agglutination patterns, respectively, were obtained. The lectins from Datura stramonium, Robinia pseudoacacia and Dolichos biflorus agglutinated isolates belonging only to Lancefield group C, being non-reactive with other isolates. These lectins were also found to be specific for "large colony type" streptococci of group C. The use of lectin agglutination in epidemiological and ecological studies of "S. milleri" is discussed.

Agglutination↗

PubMed1999

Identification and characterisation of a cytotoxic porin-lipopolysaccharide complex from Campylobacter jejuni.

A clinical isolate of Campylobacter jejuni, previously found to produce a toxin active in cell culture assays, was used for identification and characterisation of a cytotoxic porin-lipopolysaccharide (LPS) complex. This cytotoxic complex was isolated by high-performance liquid chromatography of crude concentrated culture supernate and DEAE-anion exchange chromatography. The complex had a toxic activity of 20.1 tissue culture dose50 (TCD50)/microg of protein for HEp-2 cells, 7.49 TCD50/microg of protein for HeLa cells and 1.87 TCD50/microg of protein for Chinese hamster ovary cells. Analysis by SDS-PAGE revealed a single protein band of 45 kDa and a high mol. wt carbohydrate moiety. The complex gave a positive result in the Limulus amoebocyte lysate test, indicating that the co-purifying carbohydrate was LPS, and had specificity for the lectins Galanthus nivalis agglutinin, Maackia amurensis agglutinin and Datura stramonium agglutinin. The cytotoxic activity associated with the complex was heat-labile at 70 degrees C, resistant to inactivation with trypsin and retained activity after treatment with sodium metaperiodate and the glycosidases neuraminidase and N-glycosidase F. Sequencing of the N-terminus of the protein component of the complex revealed 97% homology with the major outer-membrane porin protein from C. jejuni. The cytotoxic activity of the complex was neutralised by a polyclonal, homologous antiserum, which reacted on Western blot with the 45-kDa protein, but not by polyclonal antisera raised against a number of other bacterial toxins.

Amino Acid Sequence↗

PubMed1994

Characterization of O-glycan moieties of the 210 and 240 kDa pig intestinal receptors for Escherichia coli K88ac fimbriae.

The porcine brush border glycoproteins of 210 and 240 kDa, recognized by Escherichia coli K88ac fimbriae, contained O-linked oligosaccharides. The carbohydrate moieties were analysed by deglycosylation, lectin-binding and agglutination assays. Neuraminidase susceptibility of the 210 kDa receptor suggested that a sialoglycoprotein may act as receptor for the K88ac fimbriae. In contrast, K88ac-binding to the 210 and 240 kDa glycoproteins totally disappeared after fucosidase treatment, indicating the critical role of fucosyl residues at the receptor sites. Among the oligosaccharides extracted from these O-glycoproteins, K88ac fimbriae showed affinity for neutral sugar chains while sialylated species were not recognized. Our data suggest a possible role of the polypeptide backbone in the definition of receptor sites. Specific agglutination by K88ac-fimbriated E. coli of the erythrocytes of the hamster Mesocricetus auratus was inhibited by the anti-T peanut lectin and the lectins of Datura stramonium, Aleuria aurantia and Maackia amurensis. Hence, we propose that Gal beta 1-3GalNAc- and Fuc alpha 1-2Gal beta 1-3/4GlcNAc- are the main sequences mediating K88ac fimbrial binding. These structures were not detected in the non-adhesive piglet brush borders characterized by a high carbohydrate content. Additional oligosaccharides probably masked the underlying receptor structures.

Animals↗

PubMed1992

Putrescine N-Methyltransferase in Cultured Roots of Hyoscyamus albus: n-Butylamine as a Potent Inhibitor of the Transferase both in Vitro and in Vivo.

Biosynthesis of tropane alkaloids is thought to proceed by way of the diamine putrescine, followed by its methylation by putrescine N-methyltransferase (PMT; EC 2.1.1.53). High PMT activities were found in branch roots and/or cultured roots of several solanaceous plants. PMT was partially purified and characterized from cultured roots of Hyoscyamus albus that contain hyoscyamine as the main alkaloid. Initial velocity studies and product inhibition patterns of PMT are consistent with an ordered bi-bi mechanism, in which the K(m) values for putrescine and S-adenosyl-l-methionine are 277 and 203 mum, respectively, and the K(i) value for S-adenosyl-l-homocysteine is 110 mum. PMT efficiently N-methylated amines that have at least two amino groups separated by three or four methylene groups. Monoamines were good competitive inhibitors of PMT, among which n-butylamine, cyclohexylamine, and exo-2-aminonorbornane were most inhibitory, with respective K(i) values of 11.0, 9.1, and 10.0 mum. When n-butylamine was fed to root cultures of H. albus, the alkamine intermediates (tropinone, tropine, and pseudotropine) drastically decreased at 1 mm of the exogenous monoamine, and the hyoscyamine content decreased by 52% at 6 mm, whereas the contents of 6beta-hydroxyhyoscyamine and scopolamine did not change. Free and conjugated forms of polyamines were also measured. The n-butylamine treatment caused a large increase in the putrescine content (especially in the conjugated pool), and the spermine content also increased slightly, whereas the spermidine content decreased slightly. The increase in the putrescine pool size (approximately 40 nmol/mg dry weight) was large enough to account for the decrease in the total alkaloid pool size. Similar results were also obtained in root cultures of Datura stramonium. These studies further support the role of PMT as the first committed enzyme specific to alkaloid biosynthesis.

Journal Article↗

PubMed1993

Green Roots: Photosynthesis and Photoautotrophy in an Underground Plant Organ.

The potential for photosynthetic and photoautotrophic growth was studied in hairy root cultures of Asteraceae and Solanaceae species. Upon transfer to light, initially heterotrophic root cultures of Acmella oppositifolia and Datura innoxia greened rapidly, differentiated chloroplasts, and developed light-dependent CO2 fixation in the cortical cells. Photosynthetic potential was expressed in root cultures of all the Asteraceae genera examined (Acmella, Artemisia, Rudbeckia, Stevia, and Tagetes). Hairy roots of A. oppositifolia and D. innoxia were further adapted to photoautotrophy by growing in the presence of light and added CO2 (1-5%) and by direct or sequential transfers into media containing progressively lower sugar concentrations. The transition to photoautotrophy was accompanied by an increase in CO2 fixation and in the specific activity of 1,5-ribulose-bisphosphate carboxylase/ oxygenase (Rubisco). During the adaptation of A. oppositifolia roots to photoautotrophy, the ratio of Rubisco to phosphoenolpyruvate carboxylase increased significantly, approaching that found in the leaves. The levels and patterns of alkaloids and polyacetylenes produced by Solanaceae and Asteraceae hairy roots, respectively, were dramatically altered in photomixotrophic and photoautotrophic cultures. Photoautotrophic roots of A. oppositifolia have been mainitained in vitro for over 2 years.

Journal Article↗

PubMed1995

Partial Purification and Characterization of Hydroxycinnamoyl-Coenzyme A:Tyramine Hydroxycinnamoyltransferase from Cell Suspension Cultures of Solanum tuberosum.

A pathogen elicitor-inducible soluble acyltransferase (tyramine hydroxycinnamoyltransferase [THT], EC 2.3.1), which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-coenzyme A (CoA) esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was partially purified with a 380-fold enrichment and a 6% recovery from cell-suspension cultures of potato (Solanum tuberosum L. cv Datura). The enzyme showed specific activities of 33 mkat (kg protein)-1 (formation of feruloyltyramine). The apparent native Mr was found to be approximately 49,000. Highest activity was at pH 6.8 in K-phosphate. The isoelectric point of the enzyme was approximately pH5.2. The apparent energy of activation was calculated to be 96 kJ mol-1. The enzyme activity was stimulated more than 5-fold by 10 mM Ca2+ or Mg2+. The apparent Km values were 36 [mu]M for feruloyl-CoA and 85 and 140 [mu]M for cinnamoyl- and 4-coumaroyl-CoA, respectively. The Km value for tyramine in the presence of feruloyl-CoA was 22 [mu]M. In the presence of 4-coumaroyl-CoA, however, the Km for tyramine increased to about 230 [mu]M. The mode of action was an iso-ordered bi bi mechanism in which A, B, P, and Q equal hydroxycinnamoyl-CoA, tyramine, N-hydroxycinnamoyltyramine, and CoA, respectively. Thus, the reaction occurred in a ternary complex of the enzyme and substrates. The equilibrium constant of the reaction was determined to be 1.3 x 104. This gave a [delta]G[deg][prime] eq value of -23.5 kJ mol-1.

Journal Article↗

PubMed1972

Immunological Comparisons of Chymotrypsin Inhibitor I among Several Genera of the Solanaceae.

Microcomplement fixation was employed to compare the immunological differences that occur between purified inhibitor I from potato tubers, the four purified protomers that comprise it, and inhibitor I from tuber and leaf extracts. Total inhibitors of chymotrypsin and trypsin in leaves of seven genera of the Solanaceae were identified by enzymatic assay. In leaves of three genera, Solanum, Lycopersicum, and Datura, chymotrypsin inhibitor I was identified immunologically. In petals of all seven genera inhibitor I was also identified immunologically. With the microcomplement fixation technique inhibitor I from leaf or petal extracts of eight Solanaceae genera were compared. An immunological relationship of inhibitor I among seven of these genera was established.

Journal Article↗

PubMed1976

Variation in germination and amino Acid leakage of seeds with temperature related to membrane phase change.

Leakages of amino acids and/or fluorescent material as functions of temperature between 15 and 40 C are reported for imbibed seeds of Avena fatua L., Lactuca sativa L., Barbarea vulgaris R. Br., Amaranthus albus L., Abutilon theophrasti Medic., Lychnis alba Mill., Daucus carota L., Setaria faberi Herrm., Setaria viridis (L.) Beauv., and Datura stramonium L. The leakage indicates prominent increase in permeability of the plasmalemma in the 30 to 35 C range for 8 of the 10 kinds of seeds studied. Germination of the seeds at constant temperatures or with daily shifts in temperature is related to the membrane transition temperature for permeation by amino acids. Seeds of A. albus and A. theophrasti, which did not show membrane changes in the 25 to 40 C range, germinated best at 35 to 40 C; the other seeds germinated best below 30 C. Seeds of B. vulgaris showed rapid permeation of limiting membranes upon initial wetting with water, which was indicative of membrane disorder when dry. Leakage under anaerobiosis was observed for S. faberi seeds.

Journal Article↗

PubMed1982

Cryostorage of cloned amino Acid analog-resistant carrot and tobacco suspension cultures.

Five clones were isolated from five different amino acid analog-resistant Daucus carota L. var. Sativa and Nicotiana tabacum L. cv. Xanthi cell lines. The individual clones were similar in their resistance to dl-5-methyltryptophan, S-(2-aminoethyl)-l-cysteine, or azetidine-2-carboxylic acid, and in their corresponding free amino acid levels.The cell suspensions were stored using a controlled freezing rate at -196 degrees C with concentrations up to 40% of the four cryoprotectants: mannitol, proline, dimethylsulfoxide, glycerol, and combinations of dimethyl-sulfoxide and glycerol. No less than 55% post-thaw viability, determined by phenosafranin dye exclusion, was obtained after storage using a cryoprotectant mixture of 10% glycerol and 10% dimethylsulfoxide. Growth of the cryostored cells could be obtained consistently only by using feeder plate methodology with this combination of cryoprotectants. Post-thaw viability and percentage of cells demonstrating growth, as estimated by growth kinetics, were found to be similar. This indicates that little selection occurred during the freezing and recovery process. In addition, the amino acid analog-resistant traits were unaltered following cryostorage.Suspension cultures of Datura innoxia Mill. were frozen similarly with maximum post-thaw viability of 38%, but subsequent growth was not obtained.Protoplasts of D. innoxia, tobacco and carrot were also cryostored using a mixture of 10% dimethylsulfoxide and 10% glycerol as cryoprotectants. Viabilities of no less than 40% were obtained, however, only the carrot protoplasts regenerated cell walls and underwent cell division.

Journal Article↗

PubMed1984

6-azauracil-resistant variants of cultured plant cells lack uracil phosphoribosyltransferase activity.

6-Azauracil-resistant variants of Haplopappus gracilis (Nutt.) Gray and Datura innoxia Mill. lack activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme that catalyzes the conversion of uracil and 6-azauracil to uridine-5'-monophosphate and 6-azauridine-5'-monophosphate, respectively. Resistant cells are competent to take up uracil from their growth medium but do not convert it into a form that can be used for macromolecular synthesis. In extracts from resistant cells, orotate monophosphate decarboxylase, a target enzyme of 6-azauridine monophosphate, is fully sensitive to the phosphorylated analog. These results strongly suggest that uracil phosphoribosyltransferase is the major pathway of pyrimidine salvage in cells of these species and that loss of this enzyme activity confers on the variants resistance to 6-azauracil.

Journal Article↗

PubMed1984

Quantitation of cytokinins in biological samples using antibodies against zeatin riboside.

The cross-reactivity of antibodies elicited in rabbits against zeatin riboside, to a wide range of naturally occurring cytokinins, was examined. As well as to zeatin riboside, the antisera cross-reacted to a considerable extent with zeatin, lupinic acid, zeatin-9-glucoside, zeatin riboside 5'-monophosphate and to a much lesser, but measurable extent, with dihydrozeatin riboside and dihydrozeatin. Chromatographic methods were devised which allowed separation of all these cross-reactive compounds. Four biological samples, extracts of immature Zea mays kernels, immature seeds of Lupinus luteus, and Datura innoxia crown gall tumor tissue, and a sample of Agrobacterium tumefaciens culture supernatant, were purified by these chromatographic methods, using [(3)H]zeatin riboside as a recovery marker, and at each stage of the purification process, were subjected to radioimmunoassay over a range of dilutions. At each stage of sample purification, sample dilution curves were found to be parallel to the standard curve. Sample cytokinin levels estimated by radioimmunoassay were in close agreement to those available in the literature for similar samples assayed by alternative methods. However, in some samples, unknown cross-reacting compounds were detected.

Journal Article↗

PubMed1986

Hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, in alkaloid-producing root cultures.

Root cultures of various solanaceous plants grow well in vitro and produce large amounts of tropane alkaloids. Enzyme activity that converts hyoscyamine to 6beta-hydroxyhyoscyamine is present in cell-free extracts from cultured roots of Hyoscyamus niger L. The enzyme hyoscyamine 6beta-hydroxylase was purified 3.3-fold and characterized. The hydroxylation reaction has absolute requirements for hyoscyamine, 2-oxoglutarate, Fe(2+) ions and molecular oxygen, and ascorbate stimulates this reaction. Only the l-isomer of hyoscyamine serves as a substrate; d-hyoscyamine is nearly inactive. Comparisons were made with a number of root, shoot, and callus cultures of the Atropa, Datura, Duboisia, Hyoscyamus, and Nicotiana species for the presence of the hydroxylase activity. Decarboxylation of 2-oxoglutarate during the conversion reaction was studied using [1-(14)C]-2-oxoglutarate. A 1:1 stoichiometry was shown between the hyoscyamine-dependent formation of CO(2) from 2-oxoglutarate and the hydroxylation of hyoscyamine. Therefore, the enzyme can be classified as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). Both the supply of hyoscyamine and the hydroxylase activity determine the amounts of 6beta-hydroxyhyoscyamine and scopolamine produced in alkaloid-producing cultures.

Journal Article↗

PubMed1986

Immunochemical comparison of glutamine synthetases from some solanaceae plants.

The presence of different glutamine synthetase isoenzymes in different Solanaceae plants and their relative antigenicities against antiglutamine synthetase from tomato leaf serum were studied. All the plants tested showed one glutamine synthetase isoenzyme except for Mandragora autumnalis, which showed two, after discontinuous polyacrylamide gel electrophoresis and specific in situ assay. Antigenicities were compared by the double immunodiffusion technique. The Nicotiana glauca enzyme showed equal reactivity to that of Lycopersicon esculentum, but its antigenicity was higher than Withania frutescens, Datura stramonium, and Hyoscyamus niger. The study of relative antigenicities permitted differentiation of the glutamine synthetase enzymes from uncultivated species of Solanaceae.

Journal Article↗

PubMed1987

Epoxidation in vivo of hyoscyamine to scopolamine does not involve a dehydration step.

Hyoscyamine is epoxidized to scopolamine via 6beta-hydroxyhyoscyamine in several solanaceous plants. 6,7-Dehydrohyoscyamine has been proposed to be an intermediate in the conversion of 6beta-hydroxyhyoscyamine to scopolamine on the basis of the observation that this unsaturated alkaloid is converted to scopolamine when fed to a Datura scion. To determine whether a dehydration step is involved in scopolamine biosynthesis, [6-(18)O]6beta-hydroxyhyoscyamine was prepared from l-hyoscyamine and (18)O(2) using hyoscyamine 6beta-hydroxylase obtained from root cultures of Hyoscyamus niger L. When [6-(18)O]6beta-hydroxyhyoscyamine was fed to shoot cultures of Duboisia myoporoides R. B(R)., the labeled alkaloid was converted to scopolamine which retained (18)O in the epoxide oxygen. It is concluded that 6beta-hydroxyhyoscyamine is converted in vivo to scopolamine without a dehydration step.

Journal Article↗

PubMed1987

Transient Induction of Phenylalanine Ammonia-Lyase and 4-Coumarate: CoA Ligase mRNAs in Potato Leaves Infected with Virulent or Avirulent Races of Phytophthora infestans.

Infection of potato leaves with the fungal pathogen Phytophthora infestans (Pi) resulted in the rapid stimulation of phenylpropanoid metabolism. Increases in the activities of several mRNAs, including those encoding phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL), were detectable within a few hours postinoculation, as demonstrated by two-dimensional gel electrophoresis of proteins synthesized in vitro. This effect was closely mimicked by application of Pi culture filtrate through cut leaf stems. PAL and 4CL mRNA activities were also rapidly and transiently induced in potato cell suspension cultures by treatments with Pi culture filtrate or arachidonic acid. This induction was exploited to generate cDNA probes complementary to PAL and 4CL mRNAs. Blot hybridizations using these probes revealed almost immediate, transient and coordinate increases in the transcription rates and subsequent changes in the amounts of PAL and 4CL mRNAs in leaves treated with Pi culture filtrate. Similar changes in the mRNA amounts were found in infected leaves of potato cultivars carrying resistance genes R1 (cv Datura) or R4 (cv Isola), independent of whether a virulent or an avirulent Pi pathotype was used for inoculation. These results are discussed in relation to recent cytological observations with the same potato cultivars and Pi pathotypes.

Journal Article↗

PubMed1988

Characteristics of Five New Photoautotrophic Suspension Cultures Including Two Amaranthus Species and a Cotton Strain Growing on Ambient CO(2) Levels.

Suspension cultures of cotton (Gossypium hirsutum), Amaranthus cruentus, A. powellii, Datura innoxia, and a Nicotiana tabacum-N. glutinosa fusion hybrid were adapted to grow photoautotrophically under continuous light. The cotton strain grew with an atmosphere of ambient CO(2) (about 0.06 to 0.07% in the culture room) while the other strains required elevated CO(2) levels (5%). Photoautotrophy was indicated by the requirement for CO(2) and for light for growth. The strains grew with doubling times near 14 days and had from 50 to 600 micrograms of chlorophyll per gram of fresh weight. The cells grew in small to moderate sized clumps with cell sizes from 40 to 70 micrometers (diameter). Like most photoautotrophic cultures described so far the ribulose 1,5-bisphosphate carboxylase (RuBPcase) activity levels were well below those of mature leaves. The phosphoenolpyruvate carboxylase levels were not elevated in the C(4)Amaranthus species. The cells showed high dark respiration rates and had lower net CO(2) fixation under high O(2) conditions. Dark CO(2) fixation rates ranged from near 10 to 30% of that in light. Fluorescence emission spectra measurements show that the cell antenna pigments systems of the four strains examined are similar to that of chloroplasts of green plants. The cotton strain which was capable of growth under ambient CO(2) conditions showed the unique properties of a high RuBPcase activation level in ambient CO(2) and a stable ability to show net CO(2) fixation in 21% O(2) conditions.

Journal Article↗

PubMed1989

Photomodulation of axis extension in sparse canopies : role of the stem in the perception of light-quality signals of stand density.

A fiber optic probe inserted into plant tissues was used to investigate the effects of canopy density on the light environment in different organs. The red:far-red ratio inside the stem of Datura ferox L. seedlings and the estimated phytochrome photoequilibrium were strongly reduced by the presence of neighbors forming canopies too sparse to cause any mutual shading at the level of the leaves. In such canopies, changes in plant density had little effects on the light regime inside the leaves of the succulent Aeonium haworthii (S.D.) Webb et Berth., particularly when the lamina was kept nearly normal to the direct rays of the sun. In field experiments using D. ferox and Sinapis alba L. seedlings, the elongation of the internodes responded to various types of localized light-quality treatments that simulated different plant densities in sparse canopies. The responses were quantitatively similar to those elicited by changes in plant density. The evidence supports the hypothesis that, in stands formed by plants of similar size, the red:far-red ratio of the light that impinges laterally on the stems is among the earliest environmental cues that allow plants to detect local canopy density and adjust axis extension accordingly.

Journal Article↗

PubMed1990

Reversible accumulation of plant suspension cell cultures in g(1) phase and subsequent synchronous traverse of the cell cycle.

The induction of DNA synthesis in Datura innoxia Mill. cell cultures was determined by flow cytometry. A large fraction of the total population of cells traversed the cell cycle in synchrony when exposed to fresh medium. One hour after transfer to fresh medium, 37% of the cells were found in the process of DNA synthesis. After 24 hours of culture, 66% of the cells had accumulated in G(2) phase, and underwent cell division simultaneously. Only 10% of the cells remained in G(0) or G(1). Transfer of cells into a medium, 80% (v/v) of which was conditioned by a sister culture for 2 days, was adequate to inhibit this simultaneous traverse of the cell cycle. A large proportion of dividing cells could be arrested at the G(0) + G(1)/S boundary by exposure to 10 millimolar hydroxyurea (HU) for 12 to 24 hours. Inhibition of DNA synthesis by HU was reversible, and when resuspended into fresh culture medium synchronized cells resumed the cell cycle. Consequently, a large fraction of the cell population could be obtained in the G(2) phase. However, reversal of G(1) arrested cells was not complete and a fraction of cells did not initiate DNA synthesis. Seventy-four percent of the cells simultaneously reached 4C DNA content whereas the frequency of cells which remained in G(0) + G(1) phase was approximately 17%. Incorporation of radioactive precursors into DNA and proteins identified a population of nondividing cells which represents the fraction of cells in G(0). The frequency of cells entering G(0) was 11% at each generation. Our results indicate that almost 100% of the population of dividing cells synchronously traversed the cell cycle following suspension in fresh medium.

Journal Article↗