Evaluation of cysteine ethyl ester as efficient inducer for glutathione overproduction in Saccharomyces spp.

Economical yeast based glutathione (GSH) production is a process that is influenced by several factors like raw material and production costs, biomass production and efficient biotransformation of adequate precursors into the final product GSH. Nowadays the usage of cysteine for the microbial conversion into GSH is industrial state of practice. In the following study, the potential of different inducers to increase the GSH content was evaluated by means of design of experiments methodology. Investigations were executed in three natural Saccharomyces strains, S. cerevisiae, S. bayanus and S. boulardii, in a well suited 50 ml shake tube system. Results of shake tube experiments were confirmed in traditional baffled shake flasks and finally via batch cultivation in lab-scale bioreactors under controlled conditions. Comprehensive studies showed that the usage of cysteine ethyl ester (CEE) for the batch-wise biotransformation into GSH led up to a more than 2.2 times higher yield compared to cysteine as inducer. Additionally, the intracellular GSH content could be significantly increased for all strains in terms of 2.29 ± 0.29% for cysteine to 3.65 ± 0.23% for CEE, respectively, in bioreactors. Thus, the usage of CEE provides a highly attractive inducing strategy for the GSH overproduction.     

Restoration of Botshol (The Netherlands) by reduction of external nutrient load: recovery of a characean community,dominated by Chara connivens

Till about 1965 a rich characean community occurred in the shallow peat lake Botshol with six species of which the rare Nitellopsis obtusa and Chara major dominated at many sites. In the period 1980–1988 characean biomass strongly decreased and only two species, Chara globularis and C. connivens, remained in small populations at a few localities. Of the macrophyte Najas marina also some small populations remained, while the aquatic moss Fontinalis antipyretica and the filamentous alga Vaucheria dichotoma predominated at many sites. These phenomena may have been due to eutrophication by the input of polluted water. This process of impoverishment was stopped by restoration measures in 1989, resulting in a lower phosphorus concentration (ca 25 µg l^-1) and a higher water transparency. Immediately after these measures the Characeae community increased in abundance and number of species. During the summer of 1990, and especially 1991, a spectacular growth occurred of Chara connivens. C. connivens was often accompanied by C. major. Other species with scattered occurrence were C. aculeolata, C. aspera, C. contraria and C. globularis. The reasons for the shift in dominance from Nitellopsis obtusa to Chara connivens are discussed. From growth experiments evidence was obtained that neither the recent higher chloride level, nor the lowered phosphate concentration were the main factors for the domination of Chara connivens.     

A combined pH and temperature precipitation step facilitates the purification of tobacco-derived recombinant proteins that are sensitive to extremes of either parameter

Incubation at pH 4.0 or blanching at ∼65°C facilitates the purification of biopharmaceutical proteins from plants by precipitating most of the host cell proteins (HCPs) before chromatography. However, both methods are compatible only with pH or thermostable target proteins whereas many target proteins may irreversibly denature, e.g., at pH < 4.0. Here, we developed a combined pH/temperature treatment for clarified tobacco extracts and intact leaves. The latter were subjected to a blanching procedure, i.e., the submersion into a hot buffer. Using a design of experiments approach we identified conditions that remove ∼70% of HCPs at ∼55°C, using the thermosensitive antibody 2G12 and the pH-sensitive DsRed as model proteins. We found that pH and temperature exerted a combined effect during the precipitation of HCPs in the pH range 5.0–7.0 at 35°C–60°C. For clarified extracts, the temperature required to achieve a DsRed purity threshold of 20% total soluble protein (TSP) increased from 54°C to 63°C when the pH was increased from 6.4 to 7.3. The pH-stable antibody 2G12 was less responsive to the combined treatment, but the purity of 1% TSP was achieved at 35°C instead of 44°C when the pH was reduced from 6.3 to 5.8. When blanching intact leaves, product losses were not exacerbated at pH 4.0. Indeed, the highest DsRed purity (58% TSP) was achieved at this pH, combined with a temperature of 60°C and an incubation time of 30 min. In contrast, the highest 2G12 purity (0.7% TSP) was achieved at pH 5.1 and 40°C with an incubation time of 20 min. Our data suggest that a combined pH/temperature regime can avoid extreme values of either parameter; therefore, broadening the applicability of these simple purification techniques to other recombinant proteins.     

Comparison of selenium treatments of crops in the field

Field experiments with spring and winter barley and ryegrass were carried out to compare the effect of fertilizers enriched with selenate or selenite with foliar application on the selenium (Se) concentrations in the crops. Application of about 20 g Se/ha given as selenate or about 100 g as selenite in a PK fertilizer and about 5 g Se/ha sprayed on the plants increased the Se-concentrations from insufficient levels to levels that met the animal nutritional requirement. Ryegrass obtained sufficient Se concentrations at lower levels of added Se than did the barley. Toxic concentrations did not occur. The choice of method thus depends on the farming practice in the individual cases.     

Effect of simulated and real weightlessness on early regeneration stages of Brassica napus protoplasts

Summary Results from experiments using protoplasts in space, performed on the Biokosmos 9 satellite in 1989 and on the Space Shuttle on the IML-1-mission in 1992 and S/MM-03 in 1996, are presented. This paper focuses on the observation that the regeneration capacity of protoplasts is lower under micro-g conditions than under 1 g conditions. These aspects have been difficult to interpret and raise new questions about the mechanisms behind the observed effects. In an effort to try to find a key element to the poor regeneration capacity, ground-based studies were initiated focusing on the effect of the variable organization and quantity of corticular microtubules (CMTs) as a consequence of short periods of real and simulated weightlessness. The new results demonstrated the capacity of protoplasts to enter division, confirming the findings in space that this was affected by gravity. The percentage of dividing cells significantly decreased as a result of exposure to simulated weightlessness on a 2-D clinostat. Similar observations were made when comparing the wall components, which confirmed that the reconstitution of the cell wall was retarded under both space conditions and simulated weightlessness. The peroxidase activity in protoplasts exposed to microgravity was slightly decreased in both 0 g and 1 g flight samples compared with the ground controls, whereas activity in the protoplasts exposed to simulated weightlessness was similar to activity in the 1 g control. The observation that protoplasts had randomized and more sparse corticular microtubules when exposed to various forms of simulated and real weightlessness on a free-fall machine on the ground could indicate that the low division capacity in 0 g protoplasts was correlated with an abnormal CMT array in these protoplasts. This study has increased our knowledge of the more basic biochemical and cell biological aspects of g effects. This is an important link in preparation for the new space era, when it will be possible to follow the growth of single cells and tissue cultures for generations under microgravity conditions on the new International Space Station, which will be functional on a permanent basis from the year 2003.     

Water temperature sensitivity of EOD waveform in Brachyhypopomus pinnicaudatus

The mechanisms that trigger the onset of the breeding season depend on geographical latitude. At the edge of Gymnotiform distribution in America, variations in day length and water temperature are likely cues to initiate breeding. In this study we aim to clarify the role of temperature and the interaction between temperature and hormonal state upon electric organ discharge waveform. In breeding ponds, we measured naturally occurring changes of water temperature and of electric organ discharge waveform during two 48-h periods in a sample of identified mature males and females of Brachyhypopomus pinnicaudatus. Water temperature, day-night cycle, and sexual maturity each modified electric organ discharge waveform. Temperature sensitivity was also evaluated in the laboratory in adult sexually-differentiated individuals, adult non-differentiated fish, juveniles, and testosterone-treated fish. Our data strongly suggest an interaction between the effects of temperature and steroid hormones upon electric organ discharge waveform. High temperature (30 °C) induced a significant decay of head negative phase amplitude in temperature-sensitive fish. This sensitivity was observed in physiological conditions that coincide with low levels of steroid hormones: juveniles and adult fish kept in captivity at 20–21 °C. Conversely, temperature resistance was observed in mature fish in the breeding habitat and was induced by testosterone treatment and by captivity at 27–28 °C. Accepted: 23 May 1999     

Genetic rescue by distant trees mitigates qualitative pollen limitation imposed by fine‐scale spatial genetic structure

Restricted seed dispersal frequently leads to fine‐scale spatial genetic structure (i.e., FSGS) within plant populations. Depending on its spatial extent and the mobility of pollinators, this inflated kinship at the immediate neighbourhood can critically impoverish pollen quality. Despite the common occurrence of positive FSGS within plant populations, our knowledge regarding the role of long‐distance pollination preventing reproductive failure is still limited. Using microsatellite markers, we examined the existence of positive FSGS in two low‐density populations of the tree Pyrus bourgaeana. We also designed controlled crosses among trees differing in their kinship to investigate the effects of increased local kinship on plant reproduction. We used six pollination treatments and fully monitored fruit production, fruit and seed weight, proportion of mature seeds per fruit, and seed germination. Our results revealed positive FSGS in both study populations and lower fruit initiation in flowers pollinated with pollen from highly‐genetically related individuals within the neighbourhood, with this trend intensifying as the fruit development progressed. Besides, open‐pollinated flowers exhibited lower performance compared to those pollinated by distant pollen donors, suggesting intense qualitative pollen limitation in natural populations. We found positive fine‐scale spatial genetic structure is translated into impoverished pollen quality from nearby pollen donors which negatively impacts the reproductive success of trees in low‐density populations. Under this scenario of intrapopulation genetic rescue by distant pollen donors, the relevance of highly‐mobile pollinators for connecting spatially and genetically distant patches of trees may be crucial to safeguarding population recruitment.