Mek is a dual-specificity kinase that activates the extracellular-signal-regulated (Erk) mitogen-activated protein (MAP) kinases upon agonist binding to receptors. The Erk MAP kinase cascade is involved in cell-fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single mek gene is present in Caenorhabditis elegans, Drosophila and Xenopus, two mek homologs, Mek1 and Mek2, are present in the mammalian cascade. In the present study, we describe a mutant mouse line in which the mek1 gene has been disrupted by insertional mutagenesis. The null mutation was recessive lethal, as the homozygous mutant embryos died at 10.5 days of gestation. Histopathological analyses revealed a reduction in vascularization of the placenta that was due to a marked decrease of vascular endothelial cells in the labyrinthine region. The failure to establish a functional placenta probably explains the death of the mek1 -/embryos. Cell-migration assays indicated that mek1 -/fibroblasts could not be induced to migrate by fibronectin, although the levels of Mek2 protein and Erk activation were normal. Re-expression of Mek1 in the mutant mouse embryonic fibroblasts (MEFs) restored their ability to migrate. Our findings provide genetic evidence that establishes the unique role played by Mek1 in signal transduction. They also suggest that mek1 function is required for normal response to angiogenic signals that might promote vascularization of the labyrinthine region of the placenta.

The genomic sequence of Pseudomonas aeruginosa PAO1 was searched for the presence of open reading frames (ORFs) encoding enzymes potentially involved in the formation of Gln-tRNA and of Asn-tRNA. We found ORFs similar to known glutamyl-tRNA synthetases (GluRS), glutaminyl-tRNA synthetases (GlnRS), aspartyl-tRNA synthetases (AspRS), and trimeric tRNA-dependent amidotransferases (AdT) but none similar to known asparaginyl-tRNA synthetases (AsnRS). The absence of AsnRS was confirmed by biochemical tests with crude and fractionated extracts of P. aeruginosa PAO1, with the homologous tRNA as the substrate. The characterization of GluRS, AspRS, and AdT overproduced from their cloned genes in P. aeruginosa and purified to homogeneity revealed that GluRS is discriminating in the sense that it does not glutamylate tRNA Gln , that AspRS is nondiscriminating, and that its Asp-tRNA Asn product is transamidated by AdT. On the other hand, tRNA Gln is directly glutaminylated by GlnRS. These results show that P. aeruginosa PAO1 is the first organism known to synthesize Asn-tRNA via the indirect pathway and to synthesize Gln-tRNA via the direct pathway. The essential role of AdT in the formation of Asn-tRNA in P. aeruginosa and the absence of a similar activity in the cytoplasm of eukaryotic cells identifies AdT as a potential target for antibiotics to be designed against this human pathogen. Such novel antibiotics could be active against other multidrug-resistant gram-negative pathogens such as Burkholderia and Neisseria as well as all pathogenic gram-positive bacteria.

Hydrodynamic stresses imposed on brewing cells of Saccharomyces cerevisiae during beer processing can have a detrimental impact on beer quality. The use of centrifuges has become an efficient way to increase brewery throughput as they decrease clarification times and improve fermenter and tank conditioning efficiency. The effect of a disc stack centrifuge on yeast and beer physical stability has been investigated. In this study, a commercial ale yeast strain has been subjected to different operating conditions during centrifugation. Cell viability and intracellular pH decreased due to processing conditions encountered during yeast cropping with a centrifuge. A relationship has been established that yeast cell wall mannan, an unfilterable haze constituent, as a function of G-force and centrifugation cycles, is released from the cell wall while concurrently, particle sizes between 0.5-2.8 μm and beer haze increased. Furthermore, yeast intracellular glycogen and trehalose levels were depleted as a result of centrifugation.