Nurses' participation in the euthanasia programs of Nazi Germany.

During the Nazi era, so-called euthanasia programs were established for handicapped and mentally ill children and adults. Organized killings of an estimated 70,000 German citizens took place at killing centers and in psychiatric institutions. Nurses were active participants; they intentionally killed more than 10,000 people in these involuntary euthanasia programs. After the war was over, most of the nurses were never punished for these crimes against humanity--although some nurses were tried along with the physicians they assisted. One such trial was of 14 nurses and was held in Munich in 1965. Although some of these nurses reported that they struggled with a guilty conscience, others did not see anything wrong with their actions, and they believed that they were releasing these patients from their suffering.

2-micron vectors containing the Saccharomyces cerevisiae metallothionein gene as a selectable marker: excellent stability in complex media, and high-level expression of a recombinant protein from a CUP1-promoter-controlled expression cassette in cis.

We have constructed 2-micron-based yeast expression vectors containing a copy of the metallothionein (CUP1) gene of Saccharomyces cerevisiae as a semi-dominant, selectable marker. When used for the expression of the thrombin inhibitor hirudin, originally derived from the leech Hirudo medicinalis, these vectors displayed the following characteristics. (1) In the presence of copper salts, they were mitotically more stable than similarly designed control vectors lacking the CUP1 gene. In copper-sensitive host strains, the apparent plasmid stability was 100%, even in complex media and during fed-batch fermentation for an extended period of time. (2) Use of the CUP1-stabilized plasmids improved the production of hirudin by both copper-sensitive and copper-resistant hosts. The highest hirudin titers were obtained with a delta CUP1 host. (3) Copper selection resulted in a moderate increase in average plasmid copy numbers (up to two-fold) as assessed by measuring hirudin expression from a constitutive promoter (GAPFL). This effect was most noticeable if the vector showed an asymmetric segregation pattern (i.e., high rates of plasmid loss in the absence of copper). (4) The CUP1 marker proved particularly useful in combination with a CUP1-promoter-controlled expression cassette on the same plasmid. In such a set-up, the rates of transcription of the heterologous protein and that of the selectable marker are tightly linked. Therefore, an increase in selective pressure directly provokes an increase in product yields. In a copper-sensitive host strain, this plasmid design allowed for the production of very high amounts of biologically active hirudin. Our results clearly establish the utility of the CUP1 marker in the construction of stable yeast expression vectors.

Stable continuous constitutive expression of a heterologous protein in Saccharomyces cerevisiae without selection pressure.

The stability of heterologous protein expression in Saccharomyces cerevisiae during continuous culture without selection for plasmid-containing cells was investigated. The protein chosen was the leech thrombin inhibitor desulphato-hirudin, which is tolerated well by S. cerevisiae when over-expressed. Expression was from a 2-mu derived multicopy vector containing a synthetic hirudin gene under control of the constitutive glyceraldehyde-3-phosphate dehydrogenase derived GAPFL promoter. The behaviour of the system was studied at three dilution rates (D) corresponding to approximately 30% (0.06 h-1), 60% (0.12 h-1) and 90% (0.17 h-1) of the estimated maximum D. The level of plasmid loss was low at all Ds, with only 5-10% plasmid-free cells observed at 75 generations. The plasmid was most stably maintained at the intermediate D of 0.12 h-1, where the rate of loss was comparable to the loss of the native 2-mu plasmid. Hirudin expression was also highest at D = 0.12 h-1, possibly as a result of cell lysis at D = 0.06 h-1 and D = 0.17 h-1, leading to the release of vacuolar proteases and subsequent proteolysis of hirudin. Differences in expression levels were not a result of changes in plasmid copy number, which was in the range 40-60 throughout all three experiments. The high stability of this system at all Ds investigated shows that heterologous protein expression is not a burden to S. cerevisiae when the protein expressed is tolerated well.

Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii.

Azotobacter vinelandii was grown diazotrophically in chemostat cultures limited by sucrose, citrate, or acetate. Specific activities of cellular oxygen consumption (qO2) and nitrogenase (acetylene reduction) were measured in situ at different dilution rates (D, representing the specific growth rate mu at steady state). Sucrose-limited cultures exhibited linear relationships between qO2 and D, each of which, however, depended on the dissolved oxygen concentration in the range of 12 to 192 microM O2. From these plots, qO2 required for maintenance processes (mO2) were extrapolated. mO2 values did not increase linearly with increasing dissolved oxygen concentrations. With citrate- or acetate-limited cultures qO2 also depended on D. At 108 microM O2, however, qO2 and mO2 of the latter cultures were significantly lower than those of sucrose-limited cultures. Specific rates of acetylene reduction increased linearly with D, irrespective of the type of limitation and of the dissolved oxygen concentration (J. Kuhla and J. Oelze, Arch. Microbiol. 149:509-514, 1988). The reversible switch-off of nitrogenase activity under oxygen stress also depended on D and was independent of qO2, mO2, or the limiting substrate. Increased switch-off effects resulting from increased stress heights could be compensated for by increasing D. Since D represents not only the supply of the carbon source but also the supply of electrons and energy, the results suggest that the flux of electrons to the nitrogenase complex, rather than qO2, stabilizes nitrogenase activity against oxygen inactivation in aerobically growing A. vinelandii.

Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations.

Azotobacter vinelandii was grown diazotrophically at different dissolved oxygen concentrations (in the range of 3 to 216 microM) in sucrose-limited continuous culture. The specific nitrogenase activity, measured on the basis of acetylene reduction in situ, was dependent solely on the growth rate and was largely independent of oxygen and sucrose concentration. FeMo (Av1) and Fe (Av2) nitrogenase proteins were quantified after Western blotting (immunoblotting). When the cultures were grown at a constant dilution rate (D, representing the growth rate, mu) of 0.15.h-1, the cellular levels of both proteins were constant regardless of different dissolved oxygen concentrations. The same was true when the organisms were grown at D values above 0.15.h-1. At a lower growth rate (D = 0.09.h-1), however, and at lower oxygen concentrations cellular levels of both nitrogenase proteins were decreased. This means that catalytic activities of nitrogenase proteins were highest at low oxygen concentrations, but at higher oxygen concentrations they increased with growth rate. Under all conditions tested, however, the Av1:Av2 molar ratio was 1:(1.45 +/- 0.12). Cellular levels of flavodoxin and FeS protein II were largely constant as well. In order to estimate turnover of nitrogenase proteins in the absence of protein synthesis, chloramphenicol was added to cultures adapted to 3 and 216 microM oxygen, respectively. After 2 h of incubation, no significant decrease in the cellular levels of Av1 and Av2 could be observed. This suggests that oxygen has no significant effect on the breakdown of nitrogenase proteins.