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T H Nielsen

Publications and source records attributed to T H Nielsen.

At least 19 recordsLinked to original sources
PubMed2002

Antibiotic and biosurfactant properties of cyclic lipopeptides produced by fluorescent Pseudomonas spp. from the sugar beet rhizosphere.

Cyclic lipopeptides (CLPs) with antibiotic and biosurfactant properties are produced by a number of soil bacteria, including fluorescent Pseudomonas spp. To provide new and efficient strains for the biological control of root-pathogenic fungi in agricultural crops, we isolated approximately 600 fluorescent Pseudomonas spp. from two different agricultural soils by using three different growth media. CLP production was observed in a large proportion of the strains (approximately 60%) inhabiting the sandy soil, compared to a low proportion (approximately 6%) in the loamy soil. Chemical structure analysis revealed that all CLPs could be clustered into two major groups, each consisting of four subgroups. The two major groups varied primarily in the number of amino acids in the cyclic peptide moiety, while each of the subgroups could be differentiated by substitutions of specific amino acids in the peptide moiety. Production of specific CLPs could be affiliated with Pseudomonas fluorescens strain groups belonging to biotype I, V, or VI. In vitro analysis using both purified CLPs and whole-cell P. fluorescens preparations demonstrated that all CLPs exhibited strong biosurfactant properties and that some also had antibiotic properties towards root-pathogenic microfungi. The CLP-producing P. fluorescens strains provide a useful resource for selection of biological control agents, whether a single strain or a consortium of strains was used to maximize the synergistic effect of multiple antagonistic traits in the inoculum.

Agriculture↗
PubMed2001

N-terminal truncation affects the kinetics and structure of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana.

The enzyme fructose-6-phosphate 2-kinase (F6P,2K; 6-phosphofructo-2-kinase)/fructose-2,6-bisphosphatase (F26BPase) catalyses the formation and degradation of the regulatory metabolite fructose 2,6-bisphosphate. A cDNA encoding the bifunctional plant enzyme isolated from Arabidopsis thaliana (AtF2KP) was expressed in yeast, and the substrate affinities and allosteric properties of the affinity-purified enzyme were characterized. In addition to the known regulators 3-phosphoglycerate, dihydroxyacetone phosphate, fructose 6-phosphate and P(i), several metabolites were identified as important new effectors. PP(i), phosphoenolpyruvate and 2-phosphoglycerate strongly inhibited F6P,2K activity, whereas fructose 1,6-bisphosphate and 6-phosphogluconate inhibited F26BPase activity. Furthermore, pyruvate was an activator of F6P,2K and an inhibitor of F26BPase. Both kinase and phosphatase activities were rapidly inactivated by mild heat treatment (42 degrees C, 10 min), but the presence of phosphate protected both enzyme activities from inactivation. In addition to the catalytic regions, the Arabidopsis enzyme comprises a 345-amino-acid N-terminus of unknown function. The role of this region was examined by the expression of a series of N-terminally truncated enzymes. The full-length and truncated enzymes were analysed by gel-filtration chromatography. The full-length enzyme was eluted as a homotetramer, whereas the truncated enzymes were eluted as monomers. Deletion of the N-terminus decreased the kinase/phosphatase activity ratio by 4-fold, and decreased the affinity for the substrate fructose 6-phosphate. The data show that the N-terminus is important both for subunit assembly and for defining the kinetic properties of the enzyme.

Arabidopsis↗
PubMed2001

Cyclic lipoundecapeptide amphisin from Pseudomonas sp. strain DSS73.

The crystal structure of the lipoundecapeptide amphisin, presented here as the tetrahydrate, C(66)H(114)N(12)O(20).4H(2)O, originating from non-ribosomal biosynthesis by Pseudomonas sp. strain DSS73, has been solved to a resolution of 0.65 A. The primary structure of amphisin is beta-hydroxydecanoyl-D-Leu-D-Asp-D-allo-Thr-D-Leu-D-Leu-D-Ser-L-Leu-D-Gln-L-Leu-L-Ile-L-Asp (Leu is leucine, Asp is aspartic acid, Thr is threonine, Ser is serine, Gln is glutamine and Ile is isoleucine). The peptide is a lactone, linking Thr4 O(gamma) to the C-terminal. The stereochemistry of the beta-hydroxy acid is R. The peptide is a close analogue of the cyclic lipopeptides tensin and pholipeptin produced by Pseudomonas fluorescens. The structure of amphisin is mainly helical (3(10)-helix), with the cyclic peptide wrapping around a hydrogen-bonded water molecule. This lipopeptide is amphiphilic and has biosurfactant and antifungal properties.

Bacterial Proteins↗
PubMed2001

Tobacco transformants with strongly decreased expression of pyrophosphate:fructose-6-phosphate expression in the base of their young growing leaves contain much higher levels of fructose-2,6-bisphosphate but no major changes in fluxes.

The role of pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) in developing leaves was studied using wild-type tobacco (Nicotiana tabacum L.) and transformants with decreased expression of PFP. (i) The leaf base, which is the youngest and most actively growing area of the leaf, had 2.5-fold higher PFP activity than the leaf tip. T3 transformants, with a 56-95% decrease in PFP activity in the leaf base and an 87-97% decrease in PFP activity in the leaf tip, were obtained by selfing and re-selfing individuals from two independent transformant lines. (ii) Other enzyme activities also showed a gradient from the leaf base to the leaf tip. There was a decrease in PFK and an increase in fructose-6-phosphate,2-kinase and plastidic fructose-1, 6-bisphosphatase, whereas cytosolic fructose-1,6-bisphosphatase activity was constant. None of these gradients was altered in the transformants. (iii) Fructose-2,6-bisphosphate (Fru2,6bisP) levels were similar at the base and tip of wild-type leaves in the dark. Illumination lead to a decrease in Fru2,6bisP at the leaf tip and an increase in Fru2,6bisP at the leaf base. Compared to wild-type plants, transformants with decreased expression of PFP had up to 2-fold higher Fru2,6bisP at the leaf tip in the dark, similar levels at the leaf tip in the light, 15-fold higher levels at the leaf base in the dark, and up to 4-fold higher levels at the leaf base in the light. (iv) To investigate metabolic fluxes, leaf discs were supplied with 14CO2 in the light or [14C]glucose in the light or the dark. Discs from the leaf tip had higher rates of photosynthesis than discs from the leaf base, whereas the rate of glucose uptake and metabolism was similar in both tissues. Significantly less label was incorporated into neutral sugars, and more into anionic compounds, cell wall and protein, and amino acids in discs from the leaf base. Metabolism of 14CO2 and [14C]glucose in transformants with low PFP was similar to that in wild-type plants, except that synthesis of neutral sugars from 14CO2 was slightly reduced in discs from the base of the leaf. (v) These results reveal that the role of PFP in the growing cells in the base of the leaf differs from that in mature leaf tissue. The increase in Fru2,6bisP in the light and the high activity of PFP relative to cytosolic fructose-1,6-bisphosphatase in the base of the leaf implicate PFP in the synthesis of sucrose in the light, as well as in glycolysis. The large increase in Fru2,6bisP at the base of the leaf of transformants implies that PFP plays a more important role in metabolism at the leaf base than in mature leaf tissue. Nevertheless, there were no major changes in carbon fluxes, or leaf or plant growth in transformants with below 10% of the wild-type PFP activity at the leaf base, implying that large changes in expression can be compensated by changes in Fru2,6-bisP, even in growing tissues.

Biological Transport↗
PubMed2001

Transgenic Arabidopsis plants with decreased activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase have altered carbon partitioning.

The role of fructose-2,6-bisphosphate (Fru-2,6-P(2)) as a regulatory metabolite in photosynthetic carbohydrate metabolism was studied in transgenic Arabidopsis plants with reduced activity of Fru-6-phosphate,2-kinase/Fru-2,6-bisphosphatase. A positive correlation was observed between the Fru-6-phosphate,2-kinase activity and the level of Fru-2,6-P(2) in the leaves. The partitioning of carbon was studied by (14)CO(2) labeling of photosynthetic products. Plant lines with Fru-2,6-P(2) levels down to 5% of the levels observed in wild-type (WT) plants had significantly altered partitioning of carbon between sucrose (Suc) versus starch. The ratio of (14)C incorporated into Suc and starch increased 2- to 3-fold in the plants with low levels of Fru-2,6-P(2) compared with WT. Transgenic plant lines with intermediate levels of Fru-2,6-P(2) compared with WT had a Suc-to-starch labeling ratio similar to the WT. Levels of sugars, starch, and phosphorylated intermediates in leaves were followed during the diurnal cycle. Plants with low levels of Fru-2,6-P(2) in leaves had high levels of Suc, glucose, and Fru and low levels of triose phosphates and glucose-1-P during the light period compared with WT. During the dark period these differences were eliminated. Our data provide direct evidence that Fru-2,6-P(2) affects photosynthetic carbon partitioning in Arabidopsis. Opposed to this, Fru-2,6-P(2) does not contribute significantly to regulation of metabolite levels in darkness.

Arabidopsis↗
PubMed2000

Structure and heterologous expression of a gene encoding fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana.

A full-length cDNA clone encoding fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana (AtF2KP) was isolated. The encoded protein is composed of two different regions: (i) a 400 amino acid COOH-terminal region, covering the catalytic region of the protein which is homologous to enzymes from other eukaryotes. This region is highly conserved among plant species (88% identity to spinach F2KP). (ii) A 345 amino acid plant-specific NH(2)-terminal region, with 59% identity to spinach F2KP, which is composed of homologous motifs and intermittent variable sequences. Western blots show that F2KP from several plant species migrates in sodium dodecyl sulphate-polyacrylamide gel electrophoresis as a similar sized (93 kDa) protein. AtF2KP was expressed in Escherichia coli as a full length and a truncated (without the NH(2)-terminal region) fusion protein. Both forms had kinase as well as phosphatase activity, but presence of the NH(2)-terminal region influenced the ratio between the two activities. It is suggested that the NH(2)-terminal region represents a regulatory region, which defines specific properties of the plant enzymes. A genomic clone for the corresponding gene, AtF2KP, was isolated. The clone (9519 bp) included 23 exons, 22 introns and the promoter sequence. Southern blot analysis showed only one copy of the gene in the A. thaliana genome.

Amino Acid Sequence↗
PubMed2000

Structure, production characteristics and fungal antagonism of tensin - a new antifungal cyclic lipopeptide from Pseudomonas fluorescens strain 96.578.

AIM: To study the antagonistic activity by Pseudomonas fluorescens strain 96.578 on the plant pathogenic fungus Rhizoctonia solani. METHODS AND RESULTS: Strain 96.578 produced a new cyclic lipopeptide, tensin. High tensin production per cell was detected in liquid media with glucose, mannitol or glutamate as growth substrate while fructose, sucrose and asparagine supported low production. Tensin production was nearly constant in media with different initial C levels, while low initial N contents reduced production. When applied to sugar beet seeds, strain 96.578 produced tensin during seed germination. When challenged with strain 96.578 or purified tensin, Rhizoctonia solani reduced radial mycelium extension but increased branching and rosette formation. CONCLUSION: The antagonistic activity of strain 96.578 towards Rhizoctonia solani was caused by tensin. SIGNIFICANCE AND IMPACT OF THE STUDY: When coated onto sugar beet seeds, tensin production by strain 96.578 could be of significant importance for inhibition of mycelial growth and seed infection by Rhizoctonia solani.

Anti-Bacterial Agents↗
PubMed1999

Cloning, characterization and expression of a bifunctional fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase from potato.

We have isolated cDNA clones encoding the regulatory enzyme fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase from a potato (Solanum tuberosum) leaf cDNA library. All clones represented transcripts of the same gene (F2KP1). Functionality of the encoded protein was verified by expression of the active enzyme in Escherichia coli. The expressed enzyme had both kinase activity which forms fructose-2,6-bisphosphate from fructose-6-phosphate and ATP, and phosphatase activity which degrade fructose-2,6-bisphosphate. The recombinant potato enzyme was radiolabelled by [2-32P]fructose-2,6-bisphosphate verifying conservation of the phosphatase catalytic mechanism which involves a phospho-protein intermediate. The deduced amino acid sequence corresponding to the catalytic core for F2KPI is homologous to the fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase isolated from animals and yeast, with conservation of amino acids involved in substrate binding and catalytic mechanisms. The sequence for F2KP1 also includes a 102 amino acids long NH2-terminal with no homology to any previously identified enzymes. This NH2 terminal may be even longer since an upstream stop codon has not yet been identified. Northern blot analysis of potato showed that the F2KP1 transcript is present in several tissues including source leaves, sink leaves and flowers, whereas the transcripts were not detectable in developing tubers. Southern blot analysis of Solanum phureja suggest there to be only one copy of the gene.

Amino Acid Sequence↗
PubMed1999

Viscosinamide, a new cyclic depsipeptide with surfactant and antifungal properties produced by Pseudomonas fluorescens DR54.

Pseudomonas fluorescens DR54 showed antagonistic properties against plant pathogenic Pythium ultimum and Rhizoctonia solani both in vitro and in planta. Antifungal activity was extractable from spent growth media, and fractionation by semi-preparative HPLC resulted in isolation of an active compound, which was identified as a new bacterial cyclic lipodepsipeptide, viscosinamide, using 1D and 2D 1H-, 13C-NMR and mass spectrometry. The new antibiotic has biosurfactant properties but differs from the known biosurfactant, viscosin, by containing glutamine rather than glutamate at the amino acid position 2 (AA2). No viscosin production was observed, however, when Ps. fluorescens DR54 was cultured in media enriched with glutamate. In vitro tests showed that purified viscosinamide also reduced fungal growth and aerial mycelium development of both P. ultimum and R. solani. Viscosinamide production by Ps. fluorescens DR54 was tightly coupled to cell proliferation in the batch cultures, as the viscosinamide produced per cell mass unit approached a constant value. In batch cultures with variable initial C, N or P nutrient levels, there were no indications of elevated viscosinamide production during starvation or maintenance of the cultures in stationary phase. Analysis of cellular fractions and spent growth media showed that a major fraction of the viscosinamide produced remained bound to the cell membrane of Ps. fluorescens DR54. The isolation, determination of structure and production characteristics of the new compound with both biosurfactant and antibiotic properties have promising perspectives for the application of Ps. fluorescens DR54 in biological control.

Antifungal Agents↗
PubMed1997

Dynamics of a microbial community associated with manure hot spots as revealed by phospholipid fatty acid analyses.

Microbial community dynamics associated with manure hot spots were studied by using a model system consisting of a gel-stabilized mixture of soil and manure, placed between layers of soil, during a 3-week incubation period. The microbial biomass, measured as the total amount of phospholipid fatty acids (PLFA), had doubled within a 2-mm distance from the soil-manure interface after 3 days. Principal-component analyses demonstrated that this increase was accompanied by reproducible changes in the composition of PLFA, indicating changes in the microbial community structure. The effect of the manure was strongest in the 2-mm-thick soil layer closest to the interface, in which the PLFA composition was statistically significantly different (P < 0.05) from that of the unaffected soil layers throughout the incubation period. An effect was also observed in the soil layer 2 to 4 mm from the interface. The changes in microbial biomass and community structure were mainly attributed to the diffusion of dissolved organic carbon from the manure. During the initial period of microbial growth, PLFA, which were already more abundant in the manure than in the soil, increased in the manure core and in the 2-mm soil layer closest to the interface. After day 3, the PLFA composition of these layers gradually became more similar to that of the soil. The dynamics of individual PLFA suggested that both taxonomic and physiological changes occurred during growth. Examples of the latter were decreases in the ratios of 16:1 omega 7t to 16:1 omega 7c and of cyclopropyl fatty acids to their respective precursors, indicating a more active bacterial community. An inverse relationship between bacterial PLFA and the eucaryotic 20:4 PLFA (arachidonic acid) suggested that grazing was important.

Animals↗
PubMed1993

Lack of tolerance of headache and radial artery diameter during a 7 hour intravenous infusion of nitroglycerin.

Nitroglycerin-(NTG)-induced headache and dilatation of the radial artery were followed in a double blind, randomized, placebo-controlled, cross-over study in 6 healthy volunteers. NTG 0.5 microgram.kg-1 x min-1 or saline were infused i.v. for 7 h, and subsequently the infusion rate was doubled for 10 min. The radial artery diameter was measured repeatedly with high frequency ultrasound and pain was scored using a 10 point verbal scale. After 5 min of NTG infusion both headache and the arterial diameter differed significantly from baseline, and no further significant change occurred. The intensity of the headache was mild to medium (median headache score 3, range 1-7). The mean dilatation of the radial artery was 36%. The dilatation in each individual, was stable over time, both during NTG and placebo, and it did not change with the double infusion rate. The headache score in each individual was more fluctuant. No tolerance either to the NTG-induced headache or arterial dilatation was observed.

Adult↗
PubMed1993

Small arteries can be accurately studied in vivo, using high frequency ultrasound.

We have validated measurements of diameters of the superficial temporal artery and other small arteries in man with a newly developed 20 MHz ultrasound scanner with A, B and M-mode imaging. The diameter of a reference object was 1.202 mm vs. 1.205 mm as measured by stereomicroscopy (nonsignificant). In vitro measurements of porcine carotid arteries could be reproduced with a mean interobserver difference of 0.008 mm, and the repeatability coefficient was 0.04 mm (1.4%). The frontal branch of the human superficial temporal artery (mean 1.24 mm) was measured with intraobserver repeatability coefficients of 0.18 mm (13.8%) to 0.31 mm (23.4%). The interobserver mean difference was 0.01 mm (0.69%) and the interobserver repeatability coefficient was 0.16 mm (11.1%). Pulsatile changes of the cross sectional area of the radial plus the ulnar artery averaged 0.93 mm2 compared to 0.63 mm2 by strain-gauge plethysmography (nonsignificant). Pulsations were 4.6% in the radial artery. We conclude that high frequency ultrasound provides an accurate and reproducible measure of the diameter of small and medium sized human arteries in vivo.

Arteries↗
PubMed1993

Chelate root filling cements: biological properties.

The purpose of this study was to test in vivo and in vitro the toxicity and the antibacterial activity of an experimental chelate cement (HN cement) using zinc oxide-eugenol cement as a reference. After subcutaneous injection of the spatulated HN cement paste in rats, it induced markedly less tissue injuries than did the zinc oxide-eugenol cement. In toxicity tests using cultures of human fibroblasts, the HN cement was found to be less toxic than the reference cement. Bacteriologically, blood agar plates inoculated with Streptococcus sanguis, Staphylococcus aureus, and the anaerobic Prevotella intermedia developed inhibition zones between 3 and 12 mm upon application of both cements. Both demonstrated marked bacteriostatic and bactericidal properties.

Animals↗
PubMed1993

No effect of the 5HT2-antagonist ICI 169,369 on systolic, ergotamine-induced blood pressure changes in man.

The 5HT-antagonistic effect of drugs in man is difficult to study because of side effects to 5HT. Ergotamine, however contracts human arteries, probably by acting on 5HT receptors. This effect can be antagonized in vitro by the 5HT antagonist ICI 169,369. After an initial ergotamine challenge to select responders to ergotamine, 10 selected volunteers were given in a double blind study an oral dose of either placebo, 30 mg or 120 mg of ICI 169,369. After 2 hr an intravenous dose of 0.5 mg ergotamine tartrate was given. From 6 to 8 hr after the administration, ergotamine caused a mean decrease in the toe-arm systolic gradient, measured by strain-gauge plethysmography of 32 mmHg (P < 0.001), which was not influenced by either 30 mg or 120 mg ICI 169,369 (P < 0.4). The most likely explanation for our inability to detect any effect of ICI 169,369 on blood pressure changes, induced by ergotamine may be high binding (99%) of ICI 169,369 to plasma proteins. There was, however, indication of a per se vasodilatory effect of ICI 169,369 since the dose of 120 mg increased the toe-arm systolic gradient by 4.7 mmHg.

Administration, Oral↗
PubMed1992

Dose-dependent headache response and dilatation of limb and extracranial arteries after three doses of 5-isosorbide-mononitrate.

The aim of the present study was to compare the ability of different doses of isosorbide-5-mononitrate (5-ISMN) to cause dilatation of medium sized and small arteries, and to examine the intensity and duration of any headache produced. Ten healthy volunteers each received 3 doses of 5-ISMN and placebo on separate days. The diameters of the radial and superficial temporal arteries were repeatedly measured with high frequency ultrasound and pain was scored using a 10 point verbal scale. A clear dose-relationship was found for plasma concentrations and headache, and for changes in the diameter of the temporal artery, but not for the radial artery. It is concluded that headache after 5-ISMN is caused by arterial dilatation or by mechanisms responsible for the arterial dilatation. Ultrasound monitoring of arterial diameters is an important and sensitive tool in the evaluation of nitrates and other vasodilators.

Adult↗