Glutamine is not an essential amino acid for Sf-9 insect cells

Summary Spodoptera frugiperda (Sf-9) insect cells are fully capable of growth and proliferation in a glutamine, glutamate and aspartate-free medium, provided that ammonium ions are supplied. S. frugiperda (Sf-21) and Mamestra brassicae cells (IZD-MB-0503) also grow in glutamine-free media but not Trichoplusia ni cells (BTI-TN 5B1-4). The yield of -galactosidase in Sf-9 cells infected with a recombinant baculovirus under glutamine-free conditions was even higher than the yield obtained in glutamine containing cultures.     

Characterization of bacterial communities from activated sludge: Culture-dependent numerical identification versus in situ identification using group- and genus-specific rRNA-targeted oligonucleotide probes

The structures of bacterial communities were studied in activated sludge samples obtained from the aerobic and anaerobic zones of a wastewater treatment plant showing enhanced phosphorous removal. Samples were analyzed by in situ hybridization with oligonucleotide probes complementary to selected regions of the 16S and 23S ribosomal RNA (rRNA) characteristic for defined phylogenetic entities (genera and larger groups). The microbial community structures revealed by molecular techniques were compared with the compositions of culturable bacterial communities, obtained from the characterization of 255 isolates from tryptone-soy (TS) agar and R2A agar. These isolates were characterized by 89 physiological tests and their cellular fatty acid patterns, and identified. Culture-dependent techniques indicated the following distribution: different Aeromonas spp. (2.7–8.3% on R2A agar; 45.0–63.7% on TS agar), Acinetobacter spp. (5.4–9.0% on R2A agar; 5.0–9.1% on TS agar), Pseudomonas spp. (up to 10% on R2A agar) and Shewanella putrefaciens (up to 3.0% on R2A agar), all members of the gamma subclass of Proteobacteria, were isolated most frequently. The relatively rare isolates of the beta subclass were identified as Acidovorax spp., Alcaligenes spp., and Comamonas spp., The Gram-positive bacteria (high DNA G+C) were assigned mainly to Arthrobacter spp., Microbacterium spp., and Mycobacterium phlei. In order to assess the in situ abundance of the most frequently isolated genus, Aeromonas, two rRNA-targeted oligonucleotide probes were developed. The two gamma proteobacterial genera Aeromonas and Acinetobacter constituted less than 5% of all bacteria. In situ, Proteobacteria belonging to the beta subclass and high G+C Gram-positive bacteria were dominant. From filamentous bacteria, Sphaerotilus spp. and Leptothrix spp. could be detected occasionally. In addition, one sample contained a high proportion of the morphologically distinct filaments of Microthrix parvicella.As for the genus Acinetobacter, the relative abundance of the most frequently gamma-proteobacterial genus Aeromonas was overestimated by the intrinsic selectivity of cultivation. Cultivation on nutrient-rich medium (TS-agar) especially supported an enhanced isolation of bacteria belonging to these two genera. Correspondence to: P. Kämpfer.     

Effects of elevated CO2 and increased nitrogen deposition on photosynthesis and growth of understory plants in spruce model ecosystems

We studied the effects of atmospheric CO₂ enrichment (280, 420 and 560 l CO₂ l^–1) and increased N deposition (0,30 and 90 kg ha^–1 year^–1) on the spruce-forest understory species Oxalis acetosella, Homogyne alpina and Rubus hirtus. Clones of these species formed the ground cover in nine 0.7 m² model ecosystems with 5-year-old Picea abies trees (leaf area index of approx 2.2). Communities grew on natural forest soil in a simulated montane climate. Independently of N deposition, the rate of light-saturated net photosynthesis of leaves grown and measured at 420 l CO₂ l^–1 was higher in Oxalis and in Homogyne, but was not significantly different in Rubus compared to leaves grown and measured at the pre-industrial CO₂ concentration of 280 l l^–1. Remarkably, further CO₂ enrichment to 560 l l^–1 caused no additional increase of CO₂ uptake. With increasing CO₂ supply concentrations of non-structural carbohydrates in leaves increased and N concentrations decreased in all species, whereas N deposition had no significant effect on these traits. Above-ground biomass and leaf area production were not significantly affected by elevated CO₂ in the more vigorously growing species O. acetosella and R. hirtus, but the slow growing H. alpina produced almost twice as much biomass and 50% more leaf area per plant under 420 l CO₂ l^–1 compared to 280 l l^–1 (again no further stimulation at 560 l l^–1). In contrast, increased N addition stimulated growth in Oxalis and Rubus but had no effect on Homogyne. In Oxalis (only) biomass per plant was positively correlated with microhabitat quantum flux density at low CO₂, but not at high CO₂ indicating carbon saturation. On the other hand, the less shade-tolerant Homogyne profited from CO₂ enrichment at all understory light levels facilitating its spread into more shady micro-habitats under elevated CO₂. These species-specific responses to CO₂ and N deposition will affect community structure. The non-linear responses to elevated CO₂ of several of the traits studied here suggest that the largest responses to rising atmospheric CO₂ are under way now or have already occurred and possible future responses to further increases in CO₂ concentration are likely to be much smaller in these understory species.     

Somatic embryogenesis and organogenesis in Encephalartos dyerianus and E. natalensis

Callus cultures were initiated from zygotic embryos of Encephalartos dyerianus and E. natalensis. Callus of both species were transferred onto a modified B5 medium containing different combinations of 2,4-dichlorophenoxyacetic acid and kinetin. Somatic embryogenesis and shoot organogenesis occurred in both species. The embryos were dicotyledonary. To date none of the embryos have matured.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

The effect of ethylene, octanoic acid and a plant-derived smoke extract on the germination of light-sensitive lettuce seeds

The effects of an aqueous plant-derived smoke extract, octanoic acid and ethylene on germination of light-sensitive Grand Rapids lettuce seeds were investigated. The smoke extract brought about a concentration dependent increase in germination and a complete inhibition of germination at high concentrations. Octanoic acid could not induce germination. Ethylene at concentrations over 5 L L^–1 increased lettuce seed germination, but not to the same degree as smoke. Aqueous smoke in combination with ethylene showed a synergistic effect on germination at suboptimal smoke concentrations. At high smoke concentrations the effect of ethylene was almost completely inhibited. Octanoic acid in combination with ethylene brought about a higher level of germination than with ethylene alone, but only at the highest concentration of octanoic acid tested (1 mM). Standardized hexane and dichloromethane-partitioned smoke extracts and octanoic acid were subjected to TLC separation. The R _f -fractions in the smoke lanes showing activity in the lettuce seed bioassay did not correspond to the R _f -value of octanoic acid. As aqueous smoke can withstand autoclaving and can be separated by TLC and HPLC without loosing activity it is unlikely that the activity of aqueous smoke is linked to ethylene. It thus appears that the active compound in smoke is neither octanoic acid nor ethylene.Abbreviations TLC thin layer chromatography - HPLC High performance liquid chromatography     

Initial increase and subsequent loss of vasoactive intestinal polypeptide immunoreactivity in acetylcholinesterase-positive neurons of mouse islets transplanted to the kidney

Collagenase-isolated pancreatic islets from C57BL/6J mice were cultured overnight and transplanted under the kidney capsule of non-diabetic syngeneic hosts. Cryostat sections of grafts and fresh islets were stained for acetylcholinesterase (AChE) and vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI). Immediately after isolation, as well as 2-5 days after transplantation, VIP-LI- and AChE-positive nerve cell bodies were clearly seen in the periphery of the islets. Grafts 3-5 days old exhibited a transient and marked increase in VIP-LI nerve cell bodies and fibers. Seven days after transplantation VIP-LI nerve structures began to decrease in number and after 26-52 weeks they were no longer detectable. In contrast, AChE-positive nerve cell bodies and fibers, which showed a relatively constant pattern of distribution, were observed throughout the entire observation period. Restaining experiments demonstrated the coexistence of VIP-LI and AChE activity in the neurons. It is concluded that the grafts were extensively equipped with an intrinsic VIP-ergic and AChE-positive innervation. The initial, transient enhancement of VIP-LI expression probably reflects an adaptation of the neuro-insular complex to the preganglionic denervation, or to the ectopic environment, or both.     

Xylitol production by immobilized recombinant Saccharomyces cerevisiae in a continuous packed-bed bioreactor

Continuous xylitol production with two different immobilized recombinant Saccharomyces cerevisiae strains (H475 and S641), expressing low and high xylose reductase (XR) activities, was investigated in a lab-scale packed-bed bioreactor. The effect of hydraulic residence time (HRT; 1.3-11.3 h), substrate/cosubstrate ratio (0.5 and 1), recycling ratio (0, 5, and 10), and aeration (anaerobic and oxygen limited conditions) were studied. The cells were immobilized by gel entrapment using Ca-alginate as support and the beads were treated with Al(3+) to improve their mechanical strength. Xylose was converted to xylitol using glucose as cosubstrate for regeneration of NAD(P)H required in xylitol formation and for generation of maintenance energy. The stability of the recombinant strains after 15 days of continuous operation was evaluated by XR activity and plasmid retention analyses. Under anaerobic conditions the volumetric xylitol productivity increased with decreasing HRT with both strains. With a recycling ratio of 10, volumetric productivities as high as 3.44 and 5.80 g/L . h were obtained with the low XR strain at HRT 1.3 h and with the high XR strain at HRT 2.6 h, respectively. However, the highest overall xylitol yields on xylose and on cosubstrate were reached at higher HRTs. Lowering the xylose/cosubstrate ratio from 1 to 0.5 increased the overall yield of xylitol on xylose, but the productivity and the xylitol yield on cosubstrate decreased. Under oxygen limited conditions the effect of the recycling ratio on production parameters was masked by other factors, such as an accumulation of free cells in the bioreactor and severe genetic instability of the high XR strain. Under anaerobic conditions the instability was less severe, causing a decrease in XR activity from 0.15 to 0.10 and from 3.18 to 1.49 U/mg with the low and high XR strains, respectively. At the end of the fermentation, the fraction of plasmid bearing cells in the beads was close to 100% for the low XR strain; however, it was significantly lower for the high XR strain, particularly for cells from the interior of the beads. (c) 1996 John Wiley & Sons, Inc.