Overexpressed vs mutated Kras in murine fibroblasts: a molecular phenotyping study

Ras acts in signalling pathways regulating the activity of multiple cellular functions including cell proliferation, differentiation, and apoptosis. Amino-acid exchanges at position 12, 13, or 61 of the Kras gene convert the proto-oncogene into an activated oncogene. Until now, a direct comparison of genome-wide expression profiling studies of Kras overexpression and different Kras mutant forms in a single assay system has not been carried out. In our study, we focused on the direct comparison of global gene expression effects caused by mutations in codon 12 or 13 of the Kras gene and Kras overexpression in murine fibroblasts. We determined Kras cellular mRNA, Ras protein and activated Ras protein levels. Further, we compared our data to the proteome analysis of the same transfected cell lines. Both overexpression and mutations of Kras lead to common altered gene expression patterns. Only two genes, Lox and Col1a1, were reversely regulated in the Kras transfectants. They may contribute to the higher aggressiveness of the Kras codon 12 mutation in tumour progression. The functional annotation of differentially expressed genes revealed a high frequency of proteins involved in tumour growth and angiogenesis. These data further support the important role of these genes in tumour-associated angiogenesis

Diversity of Antibiotic-Active Bacteria Associated with the Brown Alga Laminaria saccharina from the Baltic Sea

Bacteria associated with the marine macroalga Laminaria saccharina, collected from the Kiel Fjord (Baltic Sea, Germany), were isolated and tested for antimicrobial activity. From a total of 210 isolates, 103 strains inhibited the growth of at least one microorganism from the test panel including Gram-negative and Gram-positive bacteria as well as a yeast. Most common profiles were the inhibition of Bacillus subtilis only (30%), B. subtilis and Staphylococcus lentus (25%), and B. subtilis, S. lentus, and Candida albicans (11%). In summary, the antibiotic-active isolates covered 15 different activity patterns suggesting various modes of action. On the basis of 16S rRNA gene sequence similarities >99%, 45 phylotypes were defined, which were classified into 21 genera belonging to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Phylogenetic analysis of 16S rRNA gene sequences revealed that four isolates possibly represent novel species or even genera. In conclusion, L. saccharina represents a promising source for the isolation of new bacterial taxa and antimicrobially active bacteria

Ratiometric fluorescence live imaging analysis of membrane lipid order in <em>Arabidopsis</em> mitotic cells using a lipid order-sensitive probe

SPE Pôle IPMInternational audienceEukaryotic cells contain membranes exhibiting different levels of lipid order mostly related to their relative amount of sterol-rich domains, thought to mediate temporal and spatial organization of cellular processes. We previously provided evidence in Arabidopsis thaliana that sterols are crucial for execution of cytokinesis, the last stage of cell division. Recently, we used di-4-ANEPPDHQ, a fluorescent probe sensitive to order of lipid phases, to quantify the level of membrane order of the cell plate, the membrane structure separating daughter cells during somatic cytokinesis of higher plant cells. By employing quantitative, ratiometric fluorescence microscopy for mapping localized lipid order levels, we revealed that the Arabidopsis cell plate represents a high-lipid-order domain of the plasma membrane. Here, we describe step-by-step protocols and troubleshooting for ratiometric live imaging procedures employing the di-4-ANEPPDHQ fluorescent probe for quantification of membrane lipid order during plant cell division in suspension cell cultures and roots of Arabidopsis thaliana

Recovery of supraspinal control of stepping via indirect propriospinal relay connections after spinal cord injury

Spinal cord injuries (SCIs) in humans and experimental animals are often associated with varying degrees of spontaneous functional recovery during the first months after injury. Such recovery is widely attributed to axons spared from injury that descend from the brain and bypass incomplete lesions, but its mechanisms are uncertain. To investigate the neural basis of spontaneous recovery, we used kinematic, physiological and anatomical analyses to evaluate mice with various combinations of spatially and temporally separated lateral hemisections with or without the excitotoxic ablation of intrinsic spinal cord neurons. We show that propriospinal relay connections that bypass one or more injury sites are able to mediate spontaneous functional recovery and supraspinal control of stepping, even when there has been essentially total and irreversible interruption of long descending supraspinal pathways in mice. Our findings show that pronounced functional recovery can occur after severe SCI without the maintenance or regeneration of direct projections from the brain past the lesion and can be mediated by the reorganization of descending and propriospinal connections. Targeting interventions toward augmenting the remodeling of relay connections may provide new therapeutic strategies to bypass lesions and restore function after SCI and in other conditions such as stroke and multiple sclerosis

Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis

Reactive oxygen species (ROS) are mutagenic and may thereby promote cancer. Normally, ROS levels are tightly controlled by an inducible antioxidant program that responds to cellular stressors and is predominantly regulated by the transcription factor Nrf2 (also known as Nfe2l2) and its repressor protein Keap1 (refs 2-5). In contrast to the acute physiological regulation of Nrf2, in neoplasia there is evidence for increased basal activation of Nrf2. Indeed, somatic mutations that disrupt the Nrf2-Keap1 interaction to stabilize Nrf2 and increase the constitutive transcription of Nrf2 target genes were recently identified, indicating that enhanced ROS detoxification and additional Nrf2 functions may in fact be pro-tumorigenic. Here, we investigated ROS metabolism in primary murine cells following the expression of endogenous oncogenic alleles of Kras, Braf and Myc, and found that ROS are actively suppressed by these oncogenes. K-Ras(G12D), B-Raf(V619E) and Myc(ERT2) each increased the transcription of Nrf2 to stably elevate the basal Nrf2 antioxidant program and thereby lower intracellular ROS and confer a more reduced intracellular environment. Oncogene-directed increased expression of Nrf2 is a new mechanism for the activation of the Nrf2 antioxidant program, and is evident in primary cells and tissues of mice expressing K-Ras(G12D) and B-Raf(V619E), and in human pancreatic cancer. Furthermore, genetic targeting of the Nrf2 pathway impairs K-Ras(G12D)-induced proliferation and tumorigenesis in vivo. Thus, the Nrf2 antioxidant and cellular detoxification program represents a previously unappreciated mediator of oncogenesis