Franck Amiot

Transcription of unr ( pstream of ‐ as) down‐modulates N‐ras expression in vivo

The ras proteins (Harvey, Kirsten and N‐ras) are key regulators of signal transduction and a perturbation of their GDP/GTP cycle is frequently observed in tumors. In mammals, N‐ras constitutes with unr ( pstream of ‐ as) a tightly linked tandem of ubiquitously expressed genes. Although unr and N‐ras appear to be involved in distinct functions, this unusual genetic organization could be important for the regulation of N‐ras expression. Specifically, transcription of unr could negatively regulate that of N‐ras by transcriptional interference. To investigate this possibility, we have deleted the unr promoter by homologous recombination in murine embryonic stem cells. Analysis of tissues of heterozygous mice revealed an increase in N‐ras mRNA accumulation ranging between 20 and 65%, in agreement with the suppression of a transcriptional interference.

Lethal Escherichia coli and Salmonella typhimurium endotoxemia is mediated through different pathways

Despite the differences in the molecular structure between lipopolysaccharides (LPS) isolated from Escherichia coli, Klebsiella pneumoniae or Salmonella typhimurium, the potential differences in their biological effects in vivo have not been investigated. In the present study, TNF and LT double knock‐out (TNF−/−LT−/−) mice were almost as susceptible as TNF+/+LT+/+ controls to S. typhimurium LPS, but they were significantly more resistant to lethal endotoxemia induced by E. coli or K. pneumoniae LPS. The effect was not due to endotoxin‐associated proteins. In the knock‐out mice, this difference in lethality was accompanied by decreased interleukin‐1 (IL‐1) and interferon‐γ (IFN‐γ) production after challenge with E. coli LPS, whereas after S. typhimurium LPS more IL‐1 and IFN‐γ were produced. In contrast, more IL‐10 was produced after challenge of mice with E. coli LPS than with S. typhymurium LPS. The hypothesis that a combination of pro‐inflammatory cytokines is responsible for the mortality after S. typhimurium LPS was suggested by experiments in mice deficient in IL‐1β‐converting enzyme (ICE−/− mice). ICE‐/‐mice, lacking mature IL‐1β and IL‐18, but also defective in IFN‐γ and TNF production, were completely protected against both E. coli and S. typhimurium LPS. Experiments in Toll‐like receptor (TLR)‐4 defective mice suggested that the difference is not due to differential activation of TLR4. In conclusion, TNF and LT play a central role in the lethality due to E. coli LPS, whereas the lethal effects of S. typhimurium LPS are mediated through mechanisms also involving other cytokines such as IFN‐γ, IL‐1 and IL‐18.

Secondary alveolar echinococcosis in lymphotoxin-alpha and tumour necrosis factor-alpha deficient mice: exacerbation of Echinococcus multilocularis larval growth is associated with cellular changes in the periparasitic granuloma.

The availability of mice carrying a deletion of LT‐α and tumour necrosis factor (TNF)‐α genes enabled us to investigate the role of the TNF during alveolar echinococcosis. We compared the growth rate of Echinococcus multilocularis in LT‐αTNF‐α +/+ mice to that of mice having either no or only one LT‐αTNF‐α functionnal allele. LT‐αTNF‐α−/− mice harboured a significantly higher parasite burden than did the other two populations at 5, 10, and 15 weeks of infection, and they did not survive thereafter. Liver metacestodes removed from these mice were alive and the dehydrogenase activities of peritoneal metacestodes were decreased. Liver lesions regressed in most wild‐type mice. Indeed, dead parasites were cordoned by granulomas containing numerous macrophages and lymphocytes leading to focal liver fibrosis at an early stage of infection. In contrast, most of LT‐αTNF‐α−/− mice harboured metacestodes interspersed with leucocytes, realising purulent abscesses with secondary extensive irregular fibrosis at a late stage of infection. Heterozygous mice had behavioural characteristics intermediate between homozygous mutants and wild‐type mice. Levels of E. multilocularis‐specific delayed‐type hypersensitivity and serum antibodies were slightly decreased in LT‐αTNF‐α−/− mice. This study shows that TNF‐α and/or LT‐α genes play an essential role in the immune protection mechanisms against E. multilocularis at the site of infection.

Accumulation of mature mRNA in the nuclear fraction of mammalian cells.

Little is known about the nuclear mRNA content of mammalian cells. In this study, we analyzed by Northern blotting with a panel of probes the nuclear and cytoplasmic fractions derived from several rodent cell lines. For most of the genes under study, mature mRNAs could easily be detected in the nuclear fraction and accumulated to higher levels than the corresponding precursors. In addition, significant differences in the nucleo‐cytoplasmic partition of mature mRNAs were observed between genes as well as between cell types (NIH 3T3, CTLL‐2, D3‐ES, PC‐12), indicating that this nuclear accumulation of mRNA is regulated. Thus, while it is usually considered that splicing is the limiting step of pre‐mRNA processing, these results point towards transport or nuclear retention of mRNA as a key determinant of nuclear mRNA metabolism.

Synthesis of enantiopure substituted aziridines by diastereoselective N-bromocyclization and nucleophile-mediated regioselective opening

Abstract Enantiopure β-substituted aziridines were prepared from (S)-phenylglycinol through a key N-bromocyclization of an unsaturated iminoether. A total control of the regioselectivity was observed during the opening of these aziridines by various nucleophiles (N3−, H2O, EtOH, Me2CuLi).