C-NMR analysis of Aspergillus mutants disturbed in pyruvate metabolism

The metabolic consequences of two defects in pyruvate metabolism of the hyphal fungus Aspergillus nidulans have been investigated by natural abundance ¹³C-NMR spectroscopy. A pyruvate dehydrogenase complex (pdh) mutant, grown on acetate, accumulates alanine upon starvation which is derived from mannitol reserves. The -alanine level increases further upon incubation with the non-permissive substrate -glucose. -Glutamate is absent from these spectra as it is required both for the transamination of pyruvate and as a reaction on an impaired energy metabolism in such a pdh-deficient strain. A pyruvate carboxylase (pyc) mutant, grown upon acetate, only starts to accumulate alanine after a long incubation period with -glucose, due to the long-lasting presence of phosphoenolpyruvate carboxykinase and malic enzyme, which are both induced by growth on acetate. When this strain is grown on -fructose and -glutamate, alanine also accumulates within 3 h upon transfer to -glucose.     

13C-NMR studies on the influence of pH and nitrogen source on polyol pool formation in Aspergillus nidulans

Abstract The composition of the polyol pools in Aspergillus nidulans mycelium during active growth on sucrose depends strongly on pH. At pH 2.5, only mannitol is present. A comparison between nitrate- and ammonium-grown cultures shows stimulation of the arabitol content with nitrate a former nitrogen source. When starved mycelium is incubated either with natural-abundance or ¹³C-enriched glucose, label appears rapidly in mannitol and arabitol, regardless of the nitrogen source or the pH used.     

Light-induced spectral changes of carotenoids in chromatophores of Rhodopseudomonas sphaeroides

Carotenoid difference spectra were recorded of chromatophores of Rhodopseudomonas sphaeroides energized with low light intensities versus chromatophores under dark conditions. The amplitudes of the peaks and troughs were dependent on the light intensities and the duration of illumination, but the shape of the difference spectra remained constant. Sharp isosbestic points were found at 515, 500, 481, 465, and 452 mm. When potassium-valinomycin-induced diffusion potentials (inside negative) were imposed on the chromatophores “mirror images” of the light-induced difference spectra were recorded with the same isosbestic points and the same positions but different relative amplitudes of the peaks and troughs. The results are explained in terms of changes in the shape of the vibrational splittings of the carotenoid main electronic transition. This could be the result of changes in the fluidity of the environment of the carotenoids upon energization of the membrane. Prolonged periods of illumination with low or high light intensities resulted in irreversible changes of the difference spectra. Short periods of illumination with high light intensities resulted in reversible elevations of the baseline and red shifts of peaks, troughs, and baseline crossings.     

Transglycosidase activity of Bifidobacterium adolescentis DSM 20083 α-galactosidase

Bifidobacterium adolescentis, a gram-positive saccharolytic bacterium found in the human colon, can, alongside other bacteria, utilise stachyose in vitro thanks to the production of an α-galactosidase. The enzyme was purified from the cell-free extract of Bi. adolescentis DSM 20083^T. It was found to act with retention of configuration (α→α), releasing α-galactose from p-nitrophenyl galactoside. This hydrolysis probably operates with a double-displacement mechanism, and is consistent with the observed glycosyltransferase activity. As α-galactosides are interesting substrates for bifidobacteria, we focused on the production of new types of α-galactosides using the transgalactosylation activity of Bi. adolescentisα-galactosides. Starting from melibiose, raffinose and stachyose oligosaccharides could be formed. The transferase activity was highest at pH 7 and 40 °C. Starting from 300 mM melibiose a maximum yield of 33% oligosaccharides was obtained. The oligosaccharides formed from melibiose were purified by size-exclusion chromatography and their structure was elucidated by NMR spectroscopy in combination with enzymatic degradation and sugar linkage analysis. The trisaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp and tetrasaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp were identified, and this indicates that the transgalactosylation to melibiose occurred selectively at the C-6 hydroxyl group of the galactosyl residue. The trisaccaride α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp formed could be utilised by various intestinal bacteria, including various bifidobacteria, and might be an interesting pre- and synbiotic substrate. Received: 15 March 1999 / Received revision: 8 June 1999 / Accepted: 11 June 1999     

Sucrose and starch metabolism in carrot (Daucus carota L.) cell suspensions analysed by 13-labelling: indications for a cytosol and a plastid-localized oxidative pentose phosphate pathway

Cells were grown in batch culture on a mixture of 50 mM glucose and fructose as the carbon source; either the glucose or the fructose was [1-¹³C]-labelled. In order to investigate the uptake and conversion of glucose and fructose during long-term labelling experiments in cell suspensions of Daucus carota L., samples were taken every 2 d during a 2 week culture period and sucrose and starch were assayed by means of HPLC and ¹³C-nuclear magnetic resonance. The fructose moieties of sucrose had a lower labelling percentage than the glucose moieties. Oxidative pentose phosphate pathway activity in the cytosol is suggested to be responsible for this loss of label of especially C-1 carbons. A combination of oxidative pentose phosphate pathway activity, a relatively high activity of pathway to sucrose synthesis and a slow equilibration between glucose-6-phosphate and fructose-6-phosphate could explain these results. Starch contained glucose units with a much lower labelling percentage than glucose moieties of sucrose: it was concluded that a second, plastid-localized, oxidative pentose phosphate pathway was responsible for removal of C-1 carbons of the glucosyl units used for synthesis of starch. Redistribution of label from [1-¹³C]-hexoses to [6-¹³C]-hexoses also occurred: 18-45% of the label was found at the C-6 carbons. This is a consequence of cycling between hexose phosphates and those phosphates in the cytosol catalysed by PFP. The results indicate that independent (oxidative pentose phosphate pathway mediated) sugar converting cycles exist in the cytosol and plastid.Key words: Daucus carotaL., cell suspensions, carbon-13 nuclear magnetic resonance, ¹³C-NMR, carbohydrate cycling, oxidative pentose phosphate pathway, plastid.      

Pathway of Propionate Oxidation by a Syntrophic Culture of Smithella propionica and Methanospirillum hungatei

The pathway of propionate conversion in a syntrophic coculture of Smithella propionica and Methanospirillum hungatei JF1 was investigated by ¹³C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3-¹³C]propionate was converted to [2-¹³C]acetate, with no [1-¹³C]acetate formed. Butyrate from [3-¹³C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3-¹³C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3-¹³C]propionate was also double labeled in either the C1 and C2 atoms or the C3 and C4 atoms in a ratio of about 1:1.5. Smaller amounts of single-labeled butyrate and other combinations were also produced. 1-¹³C-labeled propionate yielded both [1-¹³C]acetate and [2-¹³C]acetate. When ¹³C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above, ¹³C was never recovered in bicarbonate or methane. These results indicate that S. propionica does not degrade propionate via the methyl-malonyl-coenzyme A (CoA) pathway or any other of the known pathways, such as the acryloyl-CoA pathway or the reductive carboxylation pathway. Our results strongly suggest that propionate is dismutated to acetate and butyrate via a six-carbon intermediate.     

Integration of Life Cycle Assessment Into Agent-Based Modeling

A method is presented that allows for a life cycle assessment (LCA) to provide environmental information on an energy infrastructure system while it evolves. Energy conversion facilities are represented in an agent-based model (ABM) as distinct instances of technologies with owners capable of making decisions based on economic and environmental information. This simulation setup allows us to explore the dynamics of assembly, disassembly, and use of these systems, which typically span decades, and to analyze the effect of using LCA information in decision making.
We were able to integrate a simplified LCA into an ABM by aligning and connecting the data structures that represent the energy infrastructure and the supply chains from source to sink. By using an appropriate database containing life cycle inventory (LCI) information and by solving the scaling factors for the technology matrix, we computed the contribution to global warming in terms of carbon dioxide (CO₂) equivalents in the form of a single impact indicator for each instance of technology at each discrete simulation step. These LCAs may then serve to show each agent the impact of its activities at a global level, as indicated by its contribution to climate change. Similar to economic indicators, the LCA indicators may be fed back to the simulated decision making in the ABM to emulate the use of environmental information while the system evolves. A proof of concept was developed that is illustrated for a simplified LCA and ABM used to generate and simulate the evolution of a bioelectricity infrastructure system.     

Vegetation change in a man‐made salt marsh affected by a reduction in both grazing and drainage

Abstract. In order to restore natural salt marsh in a 460‐ha nature reserve established in man‐made salt marsh in the Dollard estuary, The Netherlands, the artificial drainage system was neglected and cattle grazing reduced. Vegetation changes were traced through two vegetation surveys and monitoring of permanent plots over 15 yr after the management had been changed. Exclosure experiments were started to distinguish grazing effects from effects of increased soil waterlogging caused by the neglect of the drainage system. Both vegetation surveys and permanent plots demonstrated a dichotomy in vegetation succession. The incidence of secondary pioneer vegetation dominated by Salicornia spp. and Suaeda maritima increased from 0 to 20%, whereas the late‐successional (Phragmites australis) vegetation from 10 to 15%. Grazing intensity decreased towards the sea. The grazed area contracted landward, which allowed vegetation dominated by tall species to increase seaward. Grazing and increased waterlogging interacted in several ways. The impact of trampling increased, and in the intensively grazed parts soil salinity increased. This can probably be explained by low vegetation cover in spring. Framework Ordination, an indirect‐gradient‐analysis technique, was used to infer the importance of environmental factors in influencing changes in species composition. Many changes were positively or negatively correlated with soil aeration and soil salinity, whereas elevation was of minor importance. Grazing accounted for only a few changes in species frequency. Changes in permanent plots were greater during the first than during the second half of the study period. In exclosures that were installed halfway through the study period, there was a relatively rapid recovery of previously dominant species that had decreased during the first half of the study period. Species richness per unit area in the reserve increased. At the seaward side of the marsh, the altered management allowed succession to proceed leading to establishment of stands of Phragmites australis, whereas on the landward side, the combination of moderate grazing with neglect of the drainage system appeared an effective measure in maintaining habitats for a wider range of halophytic species.     

The nucleotide sequence of the transforming BglII-H fragment of adenovirus type 7 DNA

The nucleotide sequence of the leftmost BglII-H fragment (0--4.5%) of weakly oncogenic human adenovirus serotype 7 (Ad7) has been determined (1568 base pairs). This is the shortest Ad7 DNA fragment reported to transform primary rat cells into an immortal cell line (Dijkema et al., 1979). The l-strand of BhlII-H was found to contain the complete information for a polypeptide of at most 28 051 daltons, followed by the putative promoter site of the next gene. Comparison of the determined Ad7 sequence with that of the corresponding region of non-oncogenic Ad5 (Van Ormondt et al., 1978; Maat and Van Ormondt, 1979) showed that the over-all organization of the two DNAs is quite similar, but that the sequences, except in regions of suspected strategic importance, diverge considerably.