Glucose as a substrate in recombinant strain fermentation technology

Summary Glucose supplements to complex growth media of Escherichia coli affect the production of a recombinant model protein under the control of a temperature-sensitive expression system. The bacterial Crabtree effect, which occurs in the presence of glucose under aerobic conditions, not only represses the formation of citric acid cycle enzymes, but also represses the formation of the plasmid-encoded product even though the synthesis of this protein is under the control of the temperature-inducible lambda P _R-promoter/cl857-repressor expression system. When the recombinant E. coli is grown at a moderate temperature (35° C) with protein hydrolysate and glucose as substrates, a biphasic growth and production pattern is observed. In the first phase, the cells grow with a high specific growth rate, utilizing glucose and forming glutamate as a byproduct. The intracellular level of recombinant protein is very low in this phase. Later, glutamate is consumed, indicating an active citric acid cycle. The degradation of glutamate is accompanied by the intracellular accumulation of high amounts of recombinant protein.     

Oxidation of ethanol by methanogenic bacteria

Methanogenium organophilum, a non-autotrophic methanogen able to use primary and secondary alcohols as hydrogen donors, was grown on ethanol. Per mol of methane formed, 2 mol of ethanol were oxidized to acetate. In crude extract, an NADP⁺-dependent alcohol dehydrogenase (ADH) with a pH optimum of about 10.0 catalyzed a rapid (5 mol/min·mg protein; 22°C) oxidation of ethanol to acetaldehyde; after prolonged incubation also acetate was detectable. With NAD⁺ only 2% of the activity was observed. F₄₂₀ was not reduced. The crude extract also contained F₄₂₀: NADP⁺ oxidoreductase (0.45 mol/min·mg protein) that was not active at the pH optimum of ADH. With added acetaldehyde no net reduction of various electron acceptors was measured. However, the acetaldehyde was dismutated to ethanol and acetate by the crude extract. The dismutation was stimulated by NADP⁺. These findings suggested that not only the dehydrogenation of alcohol but also of aldehyde to acid was coupled to NADP⁺ reduction. If the reaction was started with acetaldehyde, formed NADPH probably reduced excess aldehyde immediately to ethanol and in this way gave rise to the observed dismutation. Acetate thiokinase activity (0.11 mol/min·mg) but no acetate kinase or phosphotransacetylase activity was observed. It is concluded that during growth on ethanol further oxidation of acetaldehyde does not occur via acetylCoA and acetyl phosphate and hence is not associated with substrate level phosphorylation. The possibility exists that oxidation of both ethanol and acetaldehyde is catalyzed by ADH. Isolation of a Methanobacterium-like strain with ethanol showed that the ability to use primary alcohols also occurs in genera other than Methanogenium.Non-standard abbreviations ADH alcohol dehydrogenase - Ap₅ALi₃ P¹,P⁵-Di(adenosine-5-)pentaphosphate - DTE dithioerythritol (2,3-dihydroxy-1,4-dithiolbutane) - F₄₂₀ N-(N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-dimethyl-8-hydroxy-5-deazariboflavin-5-phosphate - Mg. Methanogenium - OD₅₇₈ optical density at 578 nm - PIPES 1,4-piperazine-diethanesulfonic acid - TRICINE N-(2-hydroxy-1,1-bis[hydroxymethyl]methyl)-glycine - Tris 2-amino-2-hydroxy-methylpropane-1,3-diol - U unit (mol substrate/min)     

Herbicide effects on planktonic systems of different complexity

Bioassays of different complexity were compared with respect to their capability to predict the environmental impact of the herbicide atrazine in aquatic systems. Acute toxicity tests with Daphnia did not yield meaningful results. Sublethal tests with Daphnia (feeding inhibition, reduction of growth and reproduction) were more sensitive, but effective concentrations of atrazine were still rather high (2 mg/L). A relatively complicated artificial food chain system that incorporated direct and indirect effects on Daphnia yielded significant reduction of daphnid population growth at 0.1 mg/L. Enclosure experiments with natural communities were by far the most sensitive tools. Community responses could be measured at concentrations as low as 1 µg/L and 0.1 µg atrazine/L. At the lowest concentration, however, communities recovered after three weeks. We conclude that in complex systems indirect effects can be more important than direct effects, so that, contrary to the conditions in simple tests, non-target organisms may be the better indicators of herbicide stress to natural communities.     

Structures of acidic O-linked polylactosaminoglycans on human skim milk mucins

O-Linked glycans were isolated from human skim milk mucins or mucin-derived high-molecular weight glycopeptides and fractionated by anion exchange chromatography into neutral and acidic alditols. Major oligosaccharides contained in the acidic fraction were purified by high performance liquid chromatography and structurally characterized by a combination of fast atom bombardment mass spectrometry, methylation analysis and 500 MHz 1H-nuclear magnetic resonance spectroscopy. The structural aspects exhibited by these major species in the acidic fraction resemble those established previously for the neutral oligosaccharides from human skim milk mucins: 1) the size of the alditols varies from tri- to decasaccharides, 2) the core structure is of the ubiquitous type 2, 3) the backbone sequences are of the poly-N-acetyllactosamine type with a particular preponderance of linearly extended GlcNAc beta(1-3)Gal (major) or GlcNAc beta(1-6)Gal units (minor).     

Attachment and multiplication,morphology and protein production of human fetal primary liver cells cultured in hormonally defined media

In Vitro Cellular &; Developmental Biology - Plant -     

Carbon and nitrogen isotope ratios in different compartments of a healthy and a declining Picea abies forest in the Fichtelgebirge,NE Bavaria

Summary Natural carbon and nitrogen isotope ratios were measured in different compartments (needles and twigs of different ages and crown positions, litter, understorey vegetation, roots and soils of different horizons) on 5 plots of a healthy and on 8 plots of a declining Norway spruce (Picea abies (L.) Karst.) forest in the Fichtelgebirge (NE Bavaria, Germany), which has recently been described in detail (Oren et al. 1988a; Schulze et al. 1989). The ¹³C values of needles did not differ between sites or change consistently with needle age, but did decrease from the sun-to the shade-crown. This result confirms earlier conclusions from gas exchange measurements that gaseous air pollutants did no long-lasting damage in an area where such damage was expected. Twigs (¹³C between-25.3 and-27.8) were significantly less depleted in ¹³C than needles (¹³C between-27.3 and-29.1), and ¹³C in twigs increased consistently with age. The ¹⁵N values of needles ranged between-2.5 and-4.1 and varied according to stand and age. In young needles ¹⁵N decreased with needle age, but remained constant or increased in needles that were 2 or 3 years old. Needles from the healthy site were more depleted in ¹⁵N than those from the declining site. The difference between sites was greater in old needles than in young ones. This differentiation presumably reflects an earlier onset of nitrogen reallocation in needles of the declining stand. ¹⁵N values in twigs were more negative than in needles (-3.5 to-5.2) and showed age- and stand-dependent trends that were similar to the needles. ¹⁵N values of roots and soil samples increased at both stands with soil depth from-3.5 in the organic layer to +4 in the mineral soil. The ¹⁵N values of roots from the mineral soil were different from those of twigs and needles. Roots from the shallower organic layer had values similar to twigs and needles. Thus, the bulk of the assimilated nitrogen was presumably taken up by the roots from the organic layer. The problem of separation of ammonium or nitrate use by roots from different soil horizons is discussed.     

The utilization of nitrogen from insect capture by different growth forms of Drosera from Southwest Australia

Summary Plants of Drosera species, neighbouring noncarnivorous plants, and arthropods on or near each Drosera sp. were collected at 11 contrasting habitat locations in SW Australia. At three of the sites clones of the rare glandless mutant form of D. erythrorhiza were collected alongside fully glandular counterparts. The ¹⁵N value (¹⁵N/¹⁴N natural isotope composition) of insect-free leaf and stem fractions was measured, and the data then used to estimate proportional dependence on insect N (%N_dI) for the respective species and growth forms of Drosera. The data indicated lower %N_dI values for rosette than for self-supporting erect or for climbing vine species. The latter two groups showed an average %N_dI value close to 50%. The %N_dI increased with length and biomass of climbing but not erect forms of Drosera. ¹⁵N values of stems were positively correlated with corresponding values for leaves of Drosera. Leaf material was on average significantly more ¹⁵N enriched than stems, possibly due to delayed transport of recent insect-derived N, or to discrimination against ¹⁵N in transfer from leaf to the rest of the plant. The comparison of ¹⁵N values of insects and arthropod prey, glandless and glandular plants of D. erythrorhiza indicated %N_dI values of 14.3, 12.2 and 32.2 at the respective sites, while matching comparisons based on ¹⁵N of insect, reference plants and glandular plants proved less definitive, with only one site recording a positive %N_dI (value of 10.4%) despite evidence at all sites of feeding on insects by the glandular plants. The use of the ¹⁵N technique for studying nutrition of carnivorous species and the ecological significance of insect feeding of different growth forms of Drosera growing in a large range of habitats is discussed.