2-Pyrrolidinone and succinimide endogenously present in several mammalian species.

2-Pyrrolidinone and succinimide were identified in blood plasma of man, rat, and mouse. Dog plasma contained only traces of 2-pyrrolidinone not exceeding significantly the detection limit of our GCMS-method. Succinimide but not 2-pyrrolidinone could also be found in the brains of rat and mouse. Evidence is presented for a metabolic pathway leading from 2-pyrrolidinone to succinimide, with 5-hydroxy-2-pyrrolidinone as an intermediate.

Effects of the novel compound aniracetam (Ro 13-5057) upon impaired learning and memory in rodents.

The effect of aniracetam (Ro 13-5057, 1-anisoyl-2-pyrrolidinone) was studied on various forms of experimentally impaired cognitive functions (learning and memory) in rodents and produced the following effects: (1) almost complete prevention of the incapacity to learn a discrete escape response in rats exposed to sublethal hypercapnia immediately before the acquisition session; (2) partial (rats) or complete (mice) prevention of the scopolamine-induced short-term amnesia for a passive avoidance task; (3) complete protection against amnesia for a passive avoidance task in rats submitted to electroconvulsive shock immediately after avoidance acquisition; (4) prevention of the long-term retention- or retrieval-deficit for a passive avoidance task induced in rats and mice by chloramphenicol or cycloheximide administered immediately after acquisition; (5) reversal, when administered as late as 1 h before the retention test, of the deficit in retention or retrieval of a passive avoidance task induced by cycloheximide injected 2 days previously; (6) prevention of the deficit in the retrieval of an active avoidance task induced in mice by subconvulsant electroshock or hypercapnia applied immediately before retrieval testing (24 h after acquisition). These improvements or normalizations of impaired cognitive functions were seen at oral aniracetam doses of 10-100 mg/kg. Generally, the dose-response curves were bell-shaped. The mechanisms underlying the activity of aniracetam and its 'therapeutic window' are unknown. Piracetam, another pyrrolidinone derivative was used for comparison. It was active only in six of nine tests and had about one-tenth the potency of aniracetam. The results indicate that aniracetam improves cognitive functions which are impaired by different procedure and in different phases of the learning and memory process.

Cyclic AMP-stimulated phosphorylation of a high molecular weight endogenous protein substrate in sub-cellular fractions of molluscan nervous system.

Cyclic AMP stimulated the incorporation of radioactive phosphate from ATP into endogenous phosphoproteins in a 20,000 X g supernatant, as well as in a 150,000 X g supernatant, prepared from Helix nervous system homogenates. No effect of cyclic AMP was observed in the respective pellet fractions. A part (but not all) of the cyclic AMP-stimulated activity in both supernatants was due to phosphorylation of an endogenous protein substrate, of apparent molecular weight 120,000 daltons as determined from its mobility in polyacrylamide gels. Little phosphorylation of 120,000 dalton material was observed in the particulate fractions, both in the presence and absence of cyclic AMP. When the 20,000 X g pellet was incubated with radioactive ATP and an exogenous calf ovary protein kinase, cyclic AMP stimulated the phosphorylation of the 120,000 dalton material. The results suggest that both membranes and cytosol from Helix nervous system contain 120,000 dalton substrates for cyclic AMP-dependent protein kinase. However, no kinase catalyzing phosphorylation of these substrates is recovered in membrane fractions following homogenization of the tissue.

Site-directed mutagenesis: effect of an extracistronic mutation on the in vitro propagation of bacteriophage Qbeta RNA.

It has been proposed that the nucleotide sequences of the 3' terminal extracistronic regions of phage RNA plus and minus strands have been strictly conserved during evolution because they are stringently required for recognition by the viral replicase. We have devised a method to generate point mutations at selected sites in the genome of phage Qbeta. The in vitro synthesis of Qbeta RAN with G leads to A transition in the 16th position from the 3' end, i.e., in the terminal extracistronic region of the genome, is outlined. When a mixture of about 60% wild-type and 40% mutant RNA was repeatedly replicated, the mutant RNA was enriched to 80%, showing that at least this point mutation in the terminal sequence of Qbeta RNA does not impair its in vitro replication, but in fact slightly accelerates it.