Cell retention-chemostat studies of hybridoma cells-analysis of hybridoma growth and metabolism in continuous suspension culture in serum-free medium

The steady-state metabolic parameters for a hybridoma cell line have been determined in continuous suspension-perfusion culture over a wide range of perfusion rates and cell bleed rates. Significant increases in viable cell concentrations and volumetric productivities were achieved at high perfusion rates and low cell bleed rates. At the low growth rates examined in this study, cellular metabolism shifted to become more oxidative, and as a result, the fraction of consumed substrate converted to inhibitory metabolic by-products was reduced. Specific antibody productivity was found to be non-growth associated. (c) 1993 John Wiley & Sons, Inc.     

Life history patterns of parental and hybrid Daphnia differ between lakes

1. We investigated whether Daphnia galeata × hyalina hybrids of Lake Constance and Lake Greifensee show the same pattern of life history parameters as previously reported for D. galeata × cucullata hybrids and whether such a pattern is consistent between Daphnia populations from those two lakes. 2. Hybrids in Lake Constance were intermediate in size compared with the parental species. Hybrids in Lake Greifensee were smaller than D. galeata. The intrinsic growth rate (r) of hybrids from Lake Constance was not significantly different from the faster growing parental taxon D. galeata. However, r of hybrids from Lake Greifensee was significantly lower than that of D. galeata. 3. The observed juvenile body length differences between the taxa varied with the clutch number. The first clutch juvenile lengths of the three taxa did not differ for Lake Constance. First clutch juveniles of Lake Greifensee D. galeata were smaller than hybrid first clutch juveniles. The third clutch juvenile length did not differ between taxa from Lake Greifensee, but D. galeata juveniles from Lake Constance were bigger than those of D. hyalina. 4. The life history pattern found in Lake Constance corresponds to previous findings from other studies. The hybrids in this lake combine the faster population growth of one parental species with a relatively small size. In the case of Lake Greifensee hybrids, the relatively large size of first clutch juveniles and the small size of the adults could be interpreted as dual adaptations to invertebrate and fish predation. We speculate that the lower population growth rate of the hybrids is a trade‐off for this twofold protection.     

Site-specific isotope fractionation of hydrogen in the biosynthesis of plant fatty acids

The overall deuterium content of plant lipids has been investigated by isotope ratio mass spectrometry (IRMS), and the site-specific natural isotope fractionation of hydrogen has been studied by ²H-NMR at natural abundance (SNIF-NMR). An analytical strategy has been developed in order to exploit the isotopomeric composition determined in clusters associated with different chemical sites of one or several fatty acid components. The method, which combines spectrometric and chromatographic data, enables isotopic criteria to be directly derived from raw vegetable oils containing in general two saturated and two unsaturated fatty acids. These results provide new information on isotopic fractionation caused by biochemical, physiological and natural environmental effects. Some alternation in the molecular deuterium distribution has been detected which may be related to the mechanism of fatty acid elongation. The successive methylene groups introduced through malonyl CoA are the subjects of different kinetic isotope effects since one of them is exclusively derived from NADH whereas the other has a contribution from pyruvate. A discriminant analysis of the cluster isotopic parameters enables several kinds of botanical precursors to be distinguished. The authenticating performances can be improved by taking into account the influence of climatic effects related to the region in which the plant grew.     

Regulation of energy metabolism in liver

Energy metabolism in liver has to cope with the special tasks of this organ in intermediary metabolism. Main ATP-generating processes in the liver cell are the respiratory chain and glycolysis, whereas main ATP-consuming processes are gluconeogenesis, urea synthesis, protein synthesis, ATPases and mitochondrial proton leak. Mitochondrial respiratory chain in the intact liver cell is subject to control mainly by substrate (hydrogen donors, ADP, oxygen) transport and supply and proton leak/slip. Whereas hormonal control is mainly on substrate supply to mitochondria, proton leak/slip is supposed to play an important role in the modulation of the efficiency of oxidative phosphorylation.     

Liming induced stimulation of the amino acid metabolism in mycorrhizal roots of Norway spruce (Picea abies [L.] Karst.)

Localization and activity of three enzymes involved in the amino acid metabolism of ectomycorrhizas were investigated within an interdisciplinary experiment performed in a mature Norway spruce stand in Southern Germany (Höglwald). The enzymes NAD-glutamate dehydrogenase and aspartate aminotransferase were present in root cells, whereas aminopeptidase was found in mycorrhizas of Norway spruce such as Piceirhiza nigra and those with the fungi Cenococcum geophilum, Elaphomyces sp., Russula ochroleuca and Tylospora sp. Mycorrhizas growing in the humus layer contained about double the amount of protein found in those taken from the upper mineral soil (0–5 cm).Acid irrigation of the soil had no effect on the activity of any of the investigated enzymes, soluble protein or total N-contents irrespective of whether roots were taken from the organic layer or from the upper mineral soil. Liming, however, stimulated the activity of the three enzymes in mycorrhizas of the organic layer (O_f+O_h) whereas it had no effect on the activity of the investigated enzymes of mycorrhizas in the upper mineral soil. This effect is attributed to increased contents of soluble organic nitrogen compounds in the soil of the limed plots as compared to the unlimed plots.     

Mammalian-heart adenylate deaminase: Cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody

A sheep antiserum against purified rabbit-heart adenylate deaminase (EC 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardiac AMPD isoform. The antiserum and the antibodies purified therefrom recognized both native and denatured rabbit-heart AMPD in immunoprecipitation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experiments further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heart AMPD revealed a major immunoreactive band with the molecular mass (81 kDa) of the soluble rabbit-heart AMPD subunit. AMPD in heart extracts from mammalian species other than rabbit (including human) was equally immunoreactive with this antiserum by quantitative immunoblot criteria. Although generally held to be in the same isoform class as heart AMPD, erythrocyte AMPD was not immunoreactive either within or across species. Nor was AMPD from most other tissues [e.g., white (gastrocnemius) muscle, lung, kidney] immunoreactive with the cardiac-directed antibody. Limited immunoreactivity was evidenced by mammalian liver, red (soleus) muscle, and brain extracts across species, indicating the presence of a minor cardiac(-like) AMPD isoform in these tissues. The results of this study characterize the tissue distribution of the cardiac AMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiac AMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochemistry.     

Detection of protein in polyacrylamide gels using an improved silver stain

A much improved silver staining procedure for the detection of protein in polyacrylamide gels of 0.8-3.0 mm thickness is described. It achieves very high sensitivity (detecting less than 0.01 ng bovine serum albumin/mm2) by overstaining and subsequently removing nonspecific background stain using a modified, reliable destaining procedure. Maximum sensitivity follows prediamine equilibration in 0.1% (w/v) formaldehyde solution. With two-dimensional electrophoresis the improved staining procedure reveals greater than 200 polypeptides in unconcentrated human urine and greater than 150 polypeptides in a single human fingerprint.     

Carbon metabolism and gas exchange in leaves of Zea mays L.

The aim of this work was to investigate the mechanism of formation of triose phosphates and 3-phosphoglycerate during photosynthetic induction in leaves of Zea mays. Simultaneous measurements of gas exchange, chlorophyll a fluorescence and metabolite contents of maize leaves were made. Leaves illuminated in the absence of CO₂ showed a build-up of triose phosphates during the first 2 min of illumination which was comparable to the build-up observed in the presence of CO₂. Isolated mesophyll protoplasts, which lack the Calvin cycle, also showed a build-up of triose phosphates upon illumination. Leaves contained amounts of phosphoglycerate mutase and enolase adequate to account for the formation of triose phosphates and 3-phosphoglycerate from intermediates of the C₄ cycle and their precursors.     

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3-position of ring B in Daucus carota takes place at the flavonoid stage.Abbreviations CHI Chalcone isomerase - CHS chalcone synthase - DMSO dimethylsulfoxide - GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase     

Some characteristics of a fast movement of auxin in intact tomato seedlings (Lycopersicon esculentum)

Using exogenous ¹⁴C-IAA, a fast movement of auxin was demonstrated in intact tomato plants ( Lycopersicon esculentum Mill, cv. Craigella) cultured in vitro. This movement is insensitive to or weakly enhanced by cold temperature (0°C) and not influenced by gravity. In short time experiments (10 min) the main part of the radioactivity which travels through the plant is IAA; in long time ones (6 h) the metabolism is much higher and leads mainly to the formation of IAAsp. The integrity of the plant is necessary for the possibility to observe such a fast movement. Isolated hypocotyl segments show only the classical polar and slow movement of auxin. Microautoradiographic techniques show that the fast movement takes place inside the plant. In the epicotyl, the vascular bundles, mainly phloem cells, are labelled; in contrast, the radioactivity detected in the hypocotyl is low, and the tracers can be visualized only inside the epidermal cells.