Novel DNA structures resulting from dTam3 excision in tobacco

A Tam3 two-element system has been designed by combining an immobilized Tam3 element with a non-autonomous dTam3 element inserted into the HPT gene. The phenotypic assay employed, restored hygromycin resistance, indicated thattrans-activation of the non-autonomous dTam3 element occurred. Molecular analyses of the excision sites revealed that the ends of the dTam3 element remain in the empty donor sites. The predominant consequence of this type of excision appears to be that excised fragments fail to re-integrate into the tobacco genome. Only one case of dTam3 re-integration could be detected. The ends of this element had been degraded upon integration into the tobacco genome. Either the altered structure of the Tam3 derivatives or tobacco host factors are influencing thetrans-activation of a dTam3 element, resulting in aberrant excision.     

Rapid acquisition of three-dimensional triple-resonance experiments using pulsed field gradient techniques

Summary A rapid method for recording three-dimensional triple-resonance experiments utilising pulsed field gradient techniques is proposed, and applied to the HNCO experiment. In order to optimise the sensitivity of the method, a short phase cycle is used in conjunction with the pulsed field gradients to select the desired coherence transfer pathway. The method is demonstrated for the HU protein.     

Liquefaction of dulse (Palmaria palmata (L.) Kuntze) by a commercial enzyme preparation and a purified endo ,β-1,4-D-xylanase

Liquefaction of dry and freshPalmaria palmata by food grade enzyme preparations and a purified endo--1,4-D-xylanase was studied.The endo--1,4-D-xylanase (EC 3.2.1.8) was purified to homogeneity from a commercial food grade enzyme prepared fromAspergillus niger. It has a molecular weight of 22 500, a pI of 3.5, is inactive toward corn arabinoxylan,p-nitrophenyl--D-xylose, carboxymethyl cellulose but shows a weak activity toward microcrystalline cellulose. It hydrolyzes oat and dulse xylan equally well in seawater and deionized water essentially into xylose and xylobiose. It is stable between pH 5.5 to 9.0 and 0 to 30 °C and its activity is optimal at pH 4.5–5.5 and 40–60 °C. It has a K_m of 2.2 and 2.8 mg ml^-1 and V_max of 3600 and 3900 nkat mg^-1 of protein on oat and dulse xylan, respectively.Acetate buffer, deionized water and seawater alone extracted 62.6 to 64.5 % of the dry weight of dry dulse, but the use of commercial food grade enzyme preparations or the purified xylanase improved liquefaction to 81.2–87.1 %. Xylose and galactose were the only sugars present in the soluble extracts. Deionized and seawater extracted 58.8–52.7 and 39.1–42.2% of the dry weight of the fresh algae collected in fall and summer, respectively. Only galactose was found in the seawater extract, while some xylose with galactose were measured in the deionized water extract of the fresh autumn algal sample. Purified and crude xylanase improved liquefaction of fresh algae to 79.8–81.4 and 71.9–77.9% of the fresh dry weight (fall and summer, respectively) in deionized and seawater, respectively, and increased the xylose content of the soluble fractions. Polysaccharides in the soluble residues were composed of 1,3/1,4-linked xylose, 1-linked galactose (floridoside) and 1,4-linked glucose (cellulose) and contained essentially 1,4-linked xylose and 1,4-linked glucose in insoluble fractions obtained after enzymatic treatment.The use of xylanase-containing food grade enzyme preparations improves liquefaction ofPalmaria palmata, particularly from fresh alga. This study indicates that processing such as drying may modify markedly the solubility ofP. palmata cell wall polysaccharides, which would imply the existence of some organization and/or other components in the fresh cell wall that lower xylan solubility in seawater.     

Partition coefficients of plant cuticles for monoterpenes

Summary Cuticle/water partition coefficients (K_c/w) for d-limonene, -pinene and -pinene were determined by an extrapolation and a desorption method. The sorption experiments were carried out with isolated angiosperm and gymnosperm cuticles and with [¹⁴C]-labelled monoterpenes, which were obtained biosynthetically. Both methods were suitable for the determination of the K_c/w of volatile hydrophobic compounds. For the angiosperm cuticles the partition coefficients are of the order of 10⁴, which indicates a high accumulation of monoterpenes in the cuticle. The values of the conifer cuticles of Picea abies (L.) Karst. and Abies alba Mill., however, are lower due to their high lignin content. This is proved by the increase of the partition coefficients after removal of polar and phenolic components. The K_c/w can be estimated with good accuracy from the octanol/water partition coefficient, which was determined experimentally.     

Dissimilatory hexaheme c nitrite reductase of “Spirillum” strain 5175: purification and properties

When grown with nitrate as terminal electron acceptor both the soluble (periplasm, cytoplasm) and the membrane fraction of Spirillum strain 5175 exhibited high nitrite reductase activity. The nitrite reductase obtained from the soluble fraction was purified 76-fold to electrophoretical homogeneity. The enzyme reduced nitrite to ammonia with a specific activity of 723 mol NO _inf2 ^sup- × (mg protein × min)^-1. The molecular mass was 58±1 kDa by SDS-PAGE compared to 59±2 kDa determined by size exclusion chromatography under nondenaturing conditions. The enzyme (as isolated) contained 5.97±0.15 heme c molecules/M_r 58 kDa. The absorption spectrum was typical for c-type cytochrome with maxima at 280, 408, 532 and 610 nm (oxidized) and at 420, 523 and 553 nm (dithionite-reduced). The enzyme (as isolated) exhibited a complex set of high-spin and lowspin ferric heme resonances with g-values at 9.82, 3,85, 3.31, 2.95, 2.30 and 1.49 in agreement with data reported for electron paramagnetic resonance spectra of nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli.Abbreviations DNRA dissimilatory nitrate reduction to ammonia - EPR electron paramagnetic resonance - PAGE polyacrylamide gel electrophoresis - NaP_i sodium phosphate - SDS sodium dodecylsulfate     

Nitrogen fixation in coniferous bark litter

Summary Non-symbiotic heterotrophic N₂ fixation in coniferous bark litter was investigated with the acetylene reduction assay under aerobic and anaerobic conditions. The litter studied was composed essentially of bark, of pH 5 and a C/N ratio of 101; the ratio of available C to available N, which governs N₂ fixation, was considerably higher. The rate of N₂ fixation was estimated as 2.5–4.4 g N. g^–1 dry wt. day^–1. Nitrogenase activity was still evident after seven months of incubation under aerobic conditions. The N₂-ase activity was O₂ dependent: under anaerobic conditions no N₂-ase activity was found unless a fermentable C source was added. The importance of N₂ fixation in N-poor litter for the maintenance of soil fertility is emphasized.     

Glycine Antagonists Structurally Related to 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol Inhibit Binding of [3H]Strychnine to Rat Brain Membranes

[3H]Strychnine binding to rat pons + medulla membranes was used as a measure of glycine receptors or glycine receptor-coupled chloride channels in vitro. A series of compounds structurally related to 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), which previously were shown to antagonize glycine responses in cat spinal cord, inhibited [3H]strychnine binding in micromolar concentrations. The most potent of these glycine antagonists, 5,6,7,8-tetrahydro-4H-isoxazolo[3,4-d]azepin-3-ol (iso-THAZ), was also the most potent inhibitor of [3H]strychnine binding, with a Ki of 1,400 nM. The Ki value for strychnine was 7.0 nM, whereas the Ki value for the mixed gamma-aminobutyric acid (GABA)/glycine antagonist 3 alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (RU 5135) was only 4.6 nM. Sodium chloride (1,000 mM) enhanced the affinity of strychnine, brucine, isostrychnine, and the nonselective GABA antagonist pitrazepin for [3H]strychnine binding sites, whereas the affinities of glycine, beta-alanine, and taurine were reduced. These sodium chloride shifts, however, were not predictive of antagonist or agonist properties, since the sodium chloride shift for the glycine antagonist iso-THAZ and of the other THIP-related antagonists were similar to those of the glycine-like agonists. The various sodium chloride shifts show that different groups of ligands bind to glycine receptor sites in different ways.