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Cell wall biosynthesis is a key target for antibacterial drugs. The major constituent of the bacterial wall, peptidoglycan, is a netlike polymer responsible for the size and shape of the cell and for resisting osmotic pressure. It consists of glycan chains of repeating disaccharide units crosslinked through short peptide chains. Peptidoglycan assembly is catalyzed by the periplasmic domain of bifunctional class A penicillin-binding proteins. Cross-linking of the peptide chains is catalyzed by their transpeptidase module, which can be inhibited by the most widely used antibiotics, the b-lactams. In contrast, no drug in clinical use inhibits the polymerization of the glycan chains, catalyzed by their glycosyltransferase module, although it is an obvious target. We report here the purification of the ectodomain of the class A penicillin-binding protein 1a from Thermotoga maritima (Tm-1a*), expressed recombinantly in Escherichia coli. A detergent screen showed that detergents with shorter aliphatic chains were better solubilizers. Cyclohexyl-hexyl-b-d-maltoside-purified Tm-1a* was found to be monomeric and to have improved thermal stability. A miniaturized, multiwell continuous fluorescence assay of the glycosyltransferase activity was used to screen for optimal reaction conditions. Tm-1a* was active as a glycosyltransferase, catalyzing the formation of glycan chains up to 16 disaccharide units long. Our results emphasize the importance of the detergent in preparing a stable monomeric ectodomain of a class A penicillin-binding protein. Our assay could be used to screen collections of compounds for inhibitors of peptidoglycan glycosyltransferases that could serve as the basis for the development of novel antibiotics.

Summary The effects of the intravenous injection of Δ9 -tetrahydrocannabinol (0.01 to 5 mg/kg body weight) on the EEG, behavior, and photically induced epilepsy, have been studied in 11 adolescent baboons (Papio papio) with marked photosensitive epilepsy. Tameness, anorexia, and akinesia were evident for 4 hr or more after 0.5 to 5 mg/kg. The EEG showed prolonged bursts of symmetrical, rhythmic spikes and waves at about 3/sec associated with no motor signs other than fixation of gaze. There was no consistent enhancement or diminution in the EEG or myoclonic response to intermittent photic stimulation after drug administration. Resume Les effets de l'injection intraveineuse de Δ9-tetrahydrocannabinol (0.01 a 5 mg/kg de poids) ont eteetudies sur l'EEG, le comportement, et l'epilepsie induite par la stimulation lumineuse intermittente chez 11 babouins adolescents, du type Papio papio, choisis pour leur haut degre de photosensibilite. Docilite, anorexie, et akinesie etaient evidentes au moins pendant les 4 heures qui suivaient une injection de 0.5 a 5 mg/kg de la drogue. L'EEG montrait la survenuede longues bouffees de pointes ondes rythmiques, symetriques a 3 C/S; celles-ci n'etaient accompagnees d'aucun signe clinique en dehors d'un regard fixe de l'animal. L'injection de drogue n'a provoque aucune augmentation ou diminution notable des reponses electro-cliniques induites par la stimulation lumineuse intermittente. Resumen Se han estudiado las modificaciones del comportamiento del EEG y de la epilepsia foto-inducida producidas por la inyeccion intravenosa de Δ9-tetrahidro-canabinol (0.01 a 5 mg/kg de peso) en 11 zambos adolescentes Papio papio con intensa epilepsia fotosensible. Se observaron evidentes signos de docilidad, anorexia y akinesia durante mas de 4 horas despues de la inyeccion. El EEG mostro brotes simetricos y prolongados de punta-onda ritmica de 3/seg sin que se detectaran signos motores a excepcion de la mirada fija. No se observo un aumento o disminucion de las respuestas mioclonicas o en el EEG a la foto-estimulacion intermitente despues de la inyeccion. Zusammenfassung Untersuchung uber die Wirkungen intravenoser Injektionen von Δ9-Tetrahydrocannabinol (0.01 bis 5 mg pro kg Korpergewicht) auf EEG, Verhalten und Fotoepilepsie bei 11 ausgewachsenen Papio papio Pavianen mit deutlicher foto-sensibler Epilepsie. Nach Injektionen von 0.5 bis 5 mg pro kg Korpergewicht zeigten die Tiere fur 4 oder mehr Stunden Zahmheit, Anorexie und Akinesie. Im EEG traten langdauernde Ausbriuche symmetrischer, rhythmischer 3/sek spikes and waves auf, die ausser einem starren Blick von keinen motorischen Symptomen begleitet waren. Die intermittierende Fotostimulation zeigte nach Injektion keine entsprechende Ver-Starkung oder Verminderung der EEG-Befunde oder eines Fotomyoklonus.