Expression of bacterial biphenyl-chlorobiphenyl dioxygenase genes in tobacco plants

Optimized plant-microbe bioremediation processes in which the plant initiates the metabolism of xenobiotics and releases the metabolites in the rhizosphere to be further degraded by the rhizobacteria is a promising alternative to restore contaminated sites in situ. However, such processes require that plants produce the metabolites that bacteria can readily oxidize. The biphenyl dioxygenase is the first enzyme of the bacterial catabolic pathway involved in the degradation of polychlorinated biphenyls. This enzyme consists of three components: the two sub-unit oxygenase (BphAE) containing a Rieske-type iron-sulfur cluster and a mononuclear iron center, the Rieske-type ferredoxin (BphF), and the FAD-containing ferredoxin reductase (BphG). In this work, based on analyses with Nicotiana benthamiana plants transiently expressing the biphenyl dioxygenase genes from Burkholderia xenovorans LB400 and transgenic Nicotiana tabacum plants transformed with each of these four genes, we have shown that each of the three biphenyl dioxygenase components can be produced individually as active protein in tobacco plants. Therefore, when BphAE, BphF, and BphG purified from plant were used to catalyze the oxygenation of 4-chlorobiphenyl, detectable amounts of 2,3-dihydro-2, 3-dihydroxy-4'-chlorobiphenyl were produced. This suggests that creating transgenic plants expressing simultaneously all four genes required to produce active biphenyl dioxygenase is feasible.     

Virulence potential and genetic diversity of Aeromonas caviae, Aeromonas veronii, and Aeromonas hydrophila clinical isolates from Mexico and Spain: a comparative study

A comparative study of 109 Aeromonas clinical isolates belonging to the 3 species most frequently isolated from patients with diarrhea in Mexico and Spain was performed to investigate the distribution of 3 prominent toxin genes and the gene encoding flagellin of lateral flagella; 4 well-established virulence factors in the genus Aeromonas. The aerolysin-hemolysin toxin genes were the most prevalent, being present in 89% of the total isolates. The ast toxin gene was conspicuously absent from the Aeromonas caviae and Aeromonas veronii groups but was present in 91% of the Aeromonas hydrophila isolates. Both the alt toxin gene and the lafA flagellin gene also had a low incidence in A. caviae and A. veronii. Differences in the prevalence of alt and lafA were observed between isolates from Mexico and Spain, confirming genus heterogeneity according to geographic location. Carriage of multiple toxin genes was primarily restricted to A. hydrophila isolates, suggesting that A. caviae and A. veronii isolates circulating in Mexico and Spain possess a limited array of virulence genes. Enterobacterial repetitive intergenetic consensus - polymerase chain reaction showed that the Aeromonas populations sampled lack dominant clones and were genetically heterogeneous, with A. caviae being the most diverse species. Further surveys of virulence determinants in genetically heterogeneous populations of Aeromonas isolates circulating worldwide are required to enhance the understanding of their capacity to cause disease.     

The Drosophila RNA-binding protein ELAV is required for commissural axon midline crossing via control of commissureless mRNA expression in neurons

Drosophila ELAV is the founding member of an evolutionarily conserved family of RNA-binding proteins considered as key inducers of neuronal differentiation. Although several ELAV-specific targets have been identified, little is known about the role of elav during neural development. Here, we report a detailed characterization of the elav mutant commissural phenotype. The reduced number of commissures in elav mutant embryos is not due to loss or misspecification of neural cells but results from defects in commissural axon projections across the midline. We establish a causal relationship between the elav mutant commissural phenotype and a reduction in the expression of commissureless, a key component of the Robo/Slit growth cone repulsive signalling pathway. In the nerve cord of elav mutant embryos, comm mRNA expression is strongly reduced in neurons, but not in midline glial cells. Furthermore, specific expression of an elav transgene in posterior neurons of each segment of an elav mutant nerve cord restores comm mRNA expression in these cells, as well as the formation of posterior commissures. Finally, forced expression of comm in specific commissural neuron subsets rescues the midline crossing defects of these neurons in elav mutant embryos, further indicating that elav acts cell autonomously on comm expression.     

Forced expression of Phox2 homeodomain transcription factors induces a branchio-visceromotor axonal phenotype

What causes motor neurons to project into the periphery is not well understood. We here show that forced expression of the homeodomain protein Phox2b, shown previously to be necessary and sufficient for branchio-visceromotor neuron development, and of its paralogue Phox2a imposes a branchiomotor-like axonal phenotype in the spinal cord. Many Phox2-transfected neurons, whose axons would normally stay within the confines of the neural tube, now project into the periphery. Once outside the neural tube, a fraction of the ectopic axons join the spinal accessory nerve, a branchiomotor nerve which, as shown here, does not develop in the absence of Phox2b. Explant studies show that the axons of Phox2-transfected neurons need attractive cues to leave the neural tube and that their outgrowth is promoted by tissues, to which branchio-visceromotor fibers normally grow. Hence, Phox2 expression is a key step in determining the peripheral axonal phenotype and thus the decision to stay within the neural tube or to project out of it.     

Spatio-temporal expression of Prospero is finely tuned to allow the correct development and function of the nervous system in Drosophila melanogaster

Adaptive animal behaviors depend upon the precise development of the nervous system that underlies them. In Drosophila melanogaster, the pan-neural prospero gene (pros), is involved in various aspects of neurogenesis including cell cycle control, axonal outgrowth, neuronal and glial cell differentiation. As these results have been generally obtained with null pros mutants inducing embryonic lethality, the role of pros during later development remains poorly known. Using several pros-Voila (prosV) alleles, that induce multiple developmental and behavioral anomalies in the larva and in adult, we explored the relationship between these phenotypes and the variation of pros expression in 5 different neural regions during pre-imaginal development. We found that the quantity of pros mRNA spliced variants and of Pros protein varied between these alleles in a tissue-specific and developmental way. Moreover, in prosV1 and prosV13 alleles, the respective decrease or increase of pros expression, affected (i) neuronal and glial cell composition, (ii) cell proliferation and death and (iii) axonal-dendritic outgrowth in a stage and cellular context dependant way. The various phenotypic consequences induced during development, related to more or less subtle differences in gene expression, indicate that Pros level needs a precise and specific adjustment in each neural organ to allow its proper function.     

Hematopoietic potential of the pre-fusion allantois

We previously showed that the fetal component of the placenta has a vigorous hematopoietic activity. Whether this organ is an environmental niche where hematopoietic stem cells (HSC) proliferate and become committed to various lineages, or whether it is also a site for HSC emergence, was left open. This issue can be addressed only if the components that will give rise to the placenta are tested prior to vascularization. The fetal part of the placenta forms through the fusion of the allantois and the chorionic plate around the stage of 7 somite pairs. The allantois, a mesodermal rudiment that provides fetal blood vessels to the placenta, was retrieved before fusion. We found in this rudiment expression of CD41, a known marker of early embryonic hematopoietic progenitors. c-Kit encoding a progenitor specific receptor was also expressed. Significantly, as early as the 1-2 somite stage, the allantois yielded erythroid, myeloid and multipotent clonogenic progenitors, when pre-cultured in toto prior to seeding in a semisolid medium. These results provide evidence that the allantois has hematopoietic potential per se. Whether this potential also involves the ability to produce HSC is still to be determined.     

CRM1-dependent nuclear export and dimerization with hMSH5 contribute to the regulation of hMSH4 subcellular localization

MSH4 and MSH5 are members of the MutS homolog family, a conserved group of proteins involved in DNA mismatch correction and homologous recombination. Although several studies have provided compelling evidences suggesting that MSH4 and MSH5 could act together in early and late stages of meiotic recombination, their precise roles are poorly understood and recent findings suggest that the human MSH4 protein may also exert a cytoplasmic function. Here we show that MSH4 is present in the cytoplasm and the nucleus of both testicular cells and transfected somatic cells. Confocal studies on transfected cells provide the first evidence that the subcellular localization of MSH4 is regulated, at least in part, by an active nuclear export pathway dependent on the exportin CRM1. We used deletion mapping and mutagenesis to define two functional nuclear export sequences within the C-terminal part of hMSH4 that mediate nuclear export through the CRM1 pathway. Our results suggest that CRM1 is also involved in MSH5 nuclear export. In addition, we demonstrate that dimerization of MSH4 and MSH5 facilitates their nuclear localization suggesting that dimerization may regulate the intracellular trafficking of these proteins. Our findings suggest that nucleocytoplasmic traffic may constitute a regulatory mechanism for MSH4 and MSH5 functions.     

Leishmania infantum: mixed T-helper-1/T-helper-2 immune response in experimentally infected BALB/c mice

The main goal of the present study was to characterise the course of infection and immunological responses developed by Leishmania infantum infected BALB/c mice. Parasite load was determined by Real-time TaqMan PCR while cytokine and Immunoglobulin G (IgG) production were assessed by ELISA. Leishmania DNA was detected in spleen and liver as soon as day 1 post-inoculation (pi) and the parasitism was sustained until the end of the experiment. The cytokine kinetics in spleen and liver was generally associated with the oscillations of parasite load. Overall, it was not observed a distinct Th1 or Th2 pattern of cytokine production during the time of experiment. The infected mice developed a mixed immune response, with concomitant production of IFN-gamma, IL-4 and IL-10, both in spleen and liver, and both IgG isotypes. However, our results suggest that, compared to liver, the spleen is more susceptible to L. infantum infection.     

Small-world networks decrease the speed of Muller's ratchet

Muller's ratchet is an evolutionary process that has been implicated in the extinction of asexual species, the evolution of non-recombining genomes, such as the mitochondria, the degeneration of the Y chromosome, and the evolution of sex and recombination. Here we study the speed of Muller's ratchet in a spatially structured population which is subdivided into many small populations (demes) connected by migration, and distributed on a graph. We studied different types of networks: regular networks (similar to the stepping-stone model), small-world networks and completely random graphs. We show that at the onset of the small-world network - which is characterized by high local connectivity among the demes but low average path length - the speed of the ratchet starts to decrease dramatically. This result is independent of the number of demes considered, but is more pronounced the larger the network and the stronger the deleterious effect of mutations. Furthermore, although the ratchet slows down with increasing migration between demes, the observed decrease in speed is smaller in the stepping-stone model than in small-world networks. As migration rate increases, the structured populations approach, but never reach, the result in the corresponding panmictic population with the same number of individuals. Since small-world networks have been shown to describe well the real contact networks among people, we discuss our results in the light of the evolution of microbes and disease epidemics.