The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N-methyl-D-aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1-R, NR2A-R and NR2B-R. The R domains were over-expressed in Escherichia coli and purified to near homogeneity. The K-d values for binding of [C-14]spermine to NR1-R, NR2A-R and NR2B-R were 19, 140, and 33 mu M, respectively. [H-3]Ifenprodil bound to NR1-R (K-d, 0.18 mu M) and NR2B-R (K-d, 0.21 mu M), but not to NR2A-R at the concentrations tested (0.1-0.8 mu M). These K-d values were confirmed by circular dichroism measurements. The K-d values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1-R and NR2A-R or NR1-R and NR2B-R was additive with that of each individual R domain. Binding of spermine to NR1-R and NR2B-R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1-R and NR2B-R are distinct.

In a speG-disrupted Escherichia coli mutant, which cannot metabolize spermidine to acetylspermidine, addition of spermidine to the medium caused a decrease in cell viability at the late stationary phase of growth. There were parallel decreases in the levels of ribosome modulation factor (RMF), the sigma(38) subunit of RNA polymerase, and the outer membrane protein C (OmpC). To clarify that these three proteins are strongly involved in cell viability, the rmf, rpoS (encoding sigma(38)), and ompC genes were disrupted. Viability of the triple mutant decreased to less than 1% of normal cells. The triple mutant had a reduced cell viability compared to any combination of double mutants, which also had a reduced cell viability. The single rmf and rpoS, but not ompC, mutant only slightly reduced cell viability. The results indicate that cooperative functions of these three proteins are necessary for cell viability at the late stationary phase. The triple mutant had a reduced level of ribosomes and of intracellular cations.

Amino acids lining the catalytic cavity of squalene–hopene cyclase of Alicyclobacillus acidocaldarius were mutated to investigate their catalytic functions. Mutagenesis of Leu607 to Lys in the central part of the cavity resulted in the production of the bicyclic γ-polypodatetraene (1) as main product, while the mutation of Phe605 to Lys near the deprotonation site of the cavity led mainly to the formation of tetracyclic 17-isodammara-20(21),24-diene (2).

The catalytic cavity of the Alicyclobacillus acidocaldarius squalene–hopene cyclase is predominantly lined by aromatic amino acids. In mutant cyclases, the four tyrosine residues in the catalytic cavity were replaced by different amino acids. The mutants showed significant differences in catalytic behavior compared to the wild‐type and to each other. Mutants Y609L, Y609C and Y609S produced the bicyclic main product γ‐polypodatetraene, while Y495L and Y612L showed a wild‐type product pattern and produced hopene as the main product. Altered product patterns were also found with Y420 mutations.

The fundamental data requirements for the authorization of plant protection products and the inclusion of active ingredients in Annex I of Council Directive 91/414/EEC (Council Directive of 15 July 1991 referring to placing plant protection products on the market (91/414/EEC). Official Journal of European Communities L 230, 19 August 1991) are described in the Annexes II and III of this Directive. Definite instructions with regard to preconditions for implementation and methodology (guidelines) concerning investigations with terrestrial plants are deficient. In addition to that, the uniform principles for the registration of plant protection products in the Member States described in Annex VI of the directive do not include any criteria concerning the risk assessment for non-target plants. However, plant protection products often show effects on non-target plants which need to be assessed as a requirement for the authorisation of the product. Hence, the German Federal Environmental Agency has developed a tiered approach to assess the effects of plant protection products on non-terrestrial plants. The risk is assessed using the effect-concentration evaluated in ecotoxicological tests and the environmental concentration predicted by validated exposure models. To protect non-target plants in terrestrial ecosystems assessment factors need to be considered. In the future, the risk for terrestrial plants needs to be addressed, also with regard to the revision of the Annexes of Directive 91/414/EEC.

Die grundlegenden Datenanforderungen für das Verfahren zur Zulassung von Pflanzenschutzmitteln und die Aufnahme von Wirkstoffen in Anhang I der Richtlinie 91/414/EWG sind in den Anhängen II und III dieser Richtlinie beschrieben. Die “einheitlichen Grundsätze” zur Bewertung der Auswirkungen von Pflanzenschutzmitteln in den EU-Mitgliedstaaten finden sich im Anhang VI der Richtlinie. Für den Prüfbereich terrestrische Arthropoden wurden diese Kriterien insbesondere im Hinblick auf die Nutzensfunktion der Arten für die landwirtschaftliche Produktion entwickelt. Defizite bestehen hinsichtlich der Prüfmethoden (Richtlinien) und der Maßstäbe zur Bewertung des Risikos für Arthropoden als integraler Bestandteil terrestrischer Biozönosen, die aufgrund der gesetzlichen Vorgaben durch den Einsatz von Pflanzenschutzmitteln nicht geschädigt werden dürfen. Das Umweltbundesamt hat zur Beurteilung der Auswirkungen auf Arthropoden ein dreistufiges Bewertungskonzept auf der Grundlage der Prüfanforderungen der Anhänge II und III der Richtlinie 91/414/EWG entwickelt. Die Risikobewertung erfolgt anhand der in Ökotoxizitätstest bestimmten Wirkkonzentrationen und der mit Hilfe validierter Modelle prognostizierten Umweltkonzentration. Zum Schutz von Arten, die nicht bekämpft werden sollen, sind Sicherheitsfaktoren einzustellen.

Die grundlegenden Datenanforderungen für das Zulassungsverfahren von Pflanzenschutzmitteln und die Aufnahme von Wirkstoffen in Anhang I der EU-Richtlinie 91/414/EWG sind in den Anhängen II und III dieser Richtlinie beschrieben. Defizite bestehen allerdings hinsichtlich konkreter Angaben über die Voraussetzungen bzw. Veranlassungen zur Durchführung und Methodik (Prüfrichtlinien) bezüglich Untersuchungen für den Bereich terrestrischer Pflanzen. Im folgenden werden daher die wissenschaftlichen Prüfanforderungen zur Bewertung der Auswirkungen von Pflanzenschutzmitteln auf terrestrische Pflanzen erläutert.