An optimized extended DNA kappa B site that enhances plasmid DNA nuclear import and gene expression.

BACKGROUND: The nuclear factor kappa B (NF kappaB) transcription factor, which shuttles between the cytoplasm and the nucleus under specific conditions, is a suitable intracellular target to increase the nuclear import of plasmid DNA. We report the design of an optimized and extended NF kappaB DNA binding sequence that promotes an efficient plasmid nuclear import. METHODS: On the basis of structural studies, the 5'-CTGGGGACTTTCCAGCTGGGGACTTTCCAGCTGGGGACTTTCCAGG-3' segment (termed 3NF) comprising three 10-bp kappaB sites (GGGACTTTCC) separated by a 5-bp optimized spacer (AGCTG) was selected for its capacity to ensure the best structural fit with NF kappaB and to fix simultaneously three proteins. Plasmids encoding luciferase and bearing this sequence (3NF-plasmids) were constructed and their nuclear import and gene expression efficiencies compared with that of plasmids containing classical kappaB motifs. RESULTS: A high luciferase expression was associated with plasmids containing one (p3NF-luc) or two (p3NF-luc-3NF) 3NF sequences. In situ hybridization experiments and quantitative measurement of the number of plasmid copies demonstrated that the nuclear delivery of 3NF-plasmids was more efficient than that of 3NF-free plasmids. Cross-linked immunoprecipitation showed that 3NF-plasmids were recognized by NF kappaB inside cells upon transfection. The nuclear delivery was inhibited with BAY 11-7085, an inhibitor of NF kappaB activation. Finally, p3NF-luc-3NF, the most efficient construct for in vitro transfection, had a long-lived luciferase expression in vivo. CONCLUSIONS: The results obtained in the present study demonstrate the NF kappaB-mediated nuclear delivery of 3NF-plasmids. Due to its high affinity for fixing several NF kappaB, the 3NF sequence is a very promising helper for a nonviral gene delivery system.

Characterization of seizures induced by acute exposure to an organophosphate herbicide, glufosinate-ammonium.

Glufosinate-ammonium (GLA), the active component of a widely used herbicide, induces convulsions in rodents and humans. In mouse, intraperitoneal treatment with 75 mg/kg GLA generates repetitive tonic-clonic seizures associated with 100% mortality within 72 h after treatment. In this context, we characterized GLA-induced seizures, their histological consequences and the effectiveness of diazepam treatment. Epileptic discharges on electroencephalographic recordings appeared simultaneously in the hippocampus and the cerebral cortex. Diazepam treatment at 6 h immediately stopped the seizures and prevented animal death. However, intermittent seizures were recorded on electroencephalogram from 6 h after diazepam treatment until 24 h, but had disappeared after 15 days. In our model, neuronal activation (c-Fos immunohistochemistry) was observed 6 h after GLA exposure in the dentate gyrus, CA1, CA3, amygdala, piriform and entorhinal cortices, indicating the activation of the limbic system. In these structures, Fluoro-Jade C and Cresyl violet staining did not show neuronal suffering. However, astroglial activation was clearly observed at 24 h and 15 days after GLA treatment in the amygdala, piriform and entorhinal cortices by PCR quantitative, western blot and immunohistochemistry. Concomitantly, glutamine synthetase mRNA expression (PCR quantitative), protein expression (western blot) and enzymatic activity were upregulated. In conclusion, our study suggests that GLA-induced seizures: (a) involved limbic structures and (b) induced astrocytosis without neuronal degeneration as an evidence of a reactive astrocyte beneficial effect for neuronal protection.

In vivo and in vitro glycogenic effects of methionine sulfoximine are different in two inbred strains of mice.

We investigated the relationship between brain glycogen anabolism and methionine sulfoximine (MSO)-induced seizures in two inbred mouse strains that presented differential susceptibility to the convulsant. CBA/J was considered a MSO-high-reactive strain and C57BL/6J a MSO-low-reactive strain. Accordingly, the dose of MSO needed to induce seizures in CBA/J mice is lower than that in C57BL/6J mice, and CBA/J mice which had seizures, died during the first convulsion. In addition, the time--course of the MSO effect is faster in CBA/J mice than that in C57BL/6J mice. Analyses were performed in C57BL/6J and CBA/J mice after administration of 75 (subconvulsive dose) and 40 mg/kg of MSO (subconvulsive dose, not lethal dose), respectively. In the preconvulsive period, MSO induced an increase in the brain glycogen content of C57BL/6J mice only. Twenty-four hours after MSO administration, the brain glycogen content increased in both strains. The activity and expression of fructose-1,6-bisphosphatase, the last key enzyme of the gluconeogenic pathway, were increased in MSO-treated C57BL/6J mice as compared to control mice, at all experimental time points, whereas they were increased in CBA/J mice only 24 h after MSO administration. These latter results correspond to CBA/J mice that did not have seizures. Interestingly, the differences observed in vivo were consistent with results in primary cultured astrocytes from the two strains. This data suggests that the metabolism impairment, which was not a consequence of seizures, could be related to the difference in seizure susceptibility between the two strains, depending on their genetic background.

Methodology for Anti-Gene Anti-IGF-I Therapy of Malignant Tumours.

The aim of this study was to establish the criteria for methodology of cellular "anti-IGF-I" therapy of malignant tumours and particularly for glioblastoma multiforme. The treatment of primary glioblastoma patients using surgery, radiotherapy, and chemotherapy was followed by subcutaneous injection of autologous cancer cells transfected by IGF-I antisense/triple helix expression vectors. The prepared cell "vaccines" should it be in the case of glioblastomas or other tumours, have shown a change of phenotype, the absence of IGF-I protein, and expression of MHC-I and B7. The peripheral blood lymphocytes, PBL cells, removed after each of two successive vaccinations, have demonstrated for all the types of tumour tested an increasing level of CD8(+) and CD8(+)28(+) molecules and a switch from CD8(+)11b(+) to CD8(+)11. All cancer patients were supervised for up to 19 months, the period corresponding to minimum survival of glioblastoma patients. The obtained results have permitted to specify the common criteria for "anti-IGF-I" strategy: characteristics sine qua non of injected "vaccines" (cloned cells IGF-I(-) and MHC-I(+)) and of PBL cells (CD8(+) increased level).