A novel, high stringency selection system allows screening of few clones for high protein expression.

To obtain highly productive mammalian cell lines, often large numbers of clones need to be screened. This is largely due to low selection stringencies, creating many, but low protein producing clones. To remedy this problem, a novel, very stringent selection system was designed, to create few, but high protein producing clones. In essence, a selection marker with a startcodon that confers attenuated translation initiation frequency was placed upstream of the gene of interest with a startcodon that confers optimal translation initiation. From the transcribed bicistronic mRNA, the selection marker is translated at a low frequency, and the protein of interest at a high frequency. This selection system is so stringent that clones form only rarely. However, application of anti-repressor elements, which increase promoter activity, did induce the formation of clones that expressed proteins at high levels. When combined with anti-repressor elements, this novel selection system can be a valuable tool to rapidly create few, but highly productive mammalian cell lines.

Diazepam-enhanced beta activity in Sturge Weber syndrome: its diagnostic significance in comparison with MRI.

OBJECTIVES: This study was performed to assess the extent of functional involvement of the affected hemisphere in Sturge Weber syndrome in comparison with the uninvolved hemisphere. To this end beta activity in the electroencephalogram (EEG) was measured, both before and after administration of diazepam intravenously (i.v.). METHODS: In 9 patients asymmetry in beta band activity was studied before and after diazepam administration. Several beta bands and asymmetry parameters were calculated. beta band asymmetries were compared with structural abnormalities (magnetic resonance imaging, MRI). RESULTS: Total beta activity was reduced in the involved hemisphere in all patients after diazepam administration. In 3 patients functional abnormalities were found in brain regions that were structurally intact. CONCLUSIONS: Decreased diazepam-enhanced beta activity in the EEG is a sensitive criterion of functional abnormality. In patients with subtle structural abnormalities diazepam-enhanced EEG may have added value in diagnosing functional involvement and in monitoring disease progression in patients.

Activation of interictal spiking in mesiotemporal lobe epilepsy by propofol-induced sleep.

The objective of this study was to test whether low-dose propofol increases the number of interictal spikes in patients with mesiotemporal lobe epilepsy, and to determine whether this is the result of intrinsic properties and is restricted to the primary epileptogenic focus. Controlled infusion of propofol in step-up/-down target concentrations of 0, 0.3, 0.6, and 0.8 mg/L was administered to 10 patients during a 3.5-hour daytime EEG registration. The number of spikes were counted and related to propofol concentration and sleep level. Results were compared with a spontaneous, nocturnal first sleep cycle in 9 of 10 patients. All patients entered nonrapid eye movement 1 sleep during propofol administration, and 8 reached nonrapid eye movement 2 sleep. In 7 patients who showed spikes, spikes were related to sleep (P < 0.05) and not to increasing (P = 0.1) or decreasing (P = 0.5) propofol concentration. Six of nine patients showed more spikes during spontaneous (nocturnal) sleep than during propofol-induced sleep. Contralateral spiking was not suppressed selectively. Low-dose propofol is a safe means of increasing spiking in these patients because it induces sleep. There were no signs of an intrinsic epileptogenicity of propofol or a selective effect on ipsilateral spikes. Controlled sleep induction will increase the yield of interictal spikes during short interictal recordings such as in magnetoencephalography.