Phylogenetic analysis of the insect order Odonata using 28S and 16S rDNA sequences: a comparison between data sets with different evolutionary rates

Molecular phylogenetic analyses were conducted for the insect order Odonata with a focus on testing the effectiveness of a slowly evolving gene to resolve deep branching and also to examine: (i) the monophyly of damselflies (the suborder Zygoptera); and (ii) the phylogenetic position of the relict dragonfly Epiophlebia superstes. Two independent molecular sources were used to reconstruct phylogeny: the 16S rRNA gene on the mitochondrial genome and the 28S rRNA gene on the nuclear genome. A comparison of the sequences showed that the obtained 28S rDNA sequences have evolved at a much slower rate than the 16S rDNA, and that the former is better than the latter for resolving deep branching in the Odonata. Both molecular sources indicated that the Zygoptera are paraphyletic, and when a reasonable weighting for among‐site rate variation was enforced for the 16S rDNA data set, E. superstes was placed between the two remaining major suborders, namely, Zygoptera and Anisoptera (dragonflies). Character reconstruction analysis suggests that multiple hits at the rapidly evolving sites in the 16S rDNA degenerated the phylogenetic signals of the data set.     

Continuous ERK activation downregulates antiproliferative genes throughout G1 phase to allow cell-cycle progression

BACKGROUND: The ERK family of MAP kinase plays a critical role in growth factor-stimulated cell-cycle progression from G0/G1 to S phase. It has been suggested that sustained activation, but not transient activation, of ERK is necessary for inducing S phase entry. Although the essential role of ERK MAP kinase in growth factor-stimulated gene expression, especially expression of immediate-early genes, is well established, it has remained unclear how ERK activity duration affects the promotion of G1 phase progression to S phase. RESULTS: We have found that inhibition of ERK activation by the MEK inhibitor or dominant-negative MEK1 even immediately before the onset of S phase leads to the cessation of S phase entry. Our analyses reveal that there are ERK-dependent downregulated genes, whose expression levels return to their original levels rapidly after ERK inactivation, and that their downregulation mostly requires AP-1 activity. Remarkably, microinjection experiments demonstrate that many of the downregulated genes act as antiproliferative genes during G1 phase and that their forced expression to the levels before growth factor stimulation even in late G1 phase blocks S phase entry. CONCLUSIONS: Thus, continuous ERK activation downregulates antiproliferative genes until the onset of S phase to allow successful G1 phase progression. This mechanism may also work as a fail-safe mechanism, which prevents inappropriate stimuli that induce transient ERK activation from causing S phase entry.     

Synthesis and anti-HCV activity of 3′,4′-oxetane nucleosides

Hepatitis C virus afflicts approximately 180 million people worldwide and currently there are no direct acting antiviral agents available to treat this disease. Our first generation nucleoside HCV inhibitor, RG7128 has already established proof-of-concept in the clinic and is currently in phase IIb clinical trials. As part of our continuing efforts to discover novel anti-HCV agents, 3′,4′-oxetane cytidine and adenosine nucleosides were prepared as inhibitors of HCV RNA replication. These nucleosides were shown not to be inhibitors of HCV as determined in a whole cell subgenomic replicon assay. However, 2′-mono/diflouro analogs, 4, 5, and 6 were readily phosphorylated to their monophosphate metabolites by deoxycytidine kinase and their triphosphate derivatives were shown to be inhibitors of HCV NS5B polymerase in vitro. Lack of anti-HCV activity in the replicon assay may be due to the inability of the monophosphates to be converted to their corresponding diphosphates.     

Horizontal transfer of non-conjugative plasmid in colony biofilm of Escherichia coli on food-based media

We previously showed that non-conjugative, non-viral lateral plasmid transfer occurs in a colony biofilm of mixed Escherichia coli strains cultured on common laboratory media, such as LB agar. In this report, to investigate the possibility of this plasmid transfer under conditions possible outside the laboratory, we examined the activities of foodstuffs and mixed food extracts, which are possible nutrients for bacteria in human environments, for supporting lateral plasmid transfer. Lateral plasmid transfer occurred in colony biofilms grown on several foodstuffs (roasted meats) and on agar media containing mixed food extracts, which consisted of sugar, milk, and extracts of several foodstuffs (vegetables, fruits, and meats). Lateral plasmid transfer did not occur in liquid culture consisting of the same mixed food extracts, suggesting the importance of colony-biofilm formation. These results suggest the possibility that lateral transfer of non-conjugative plasmid between bacterial cells occurs in biofilms grown with foods or food-like nutrients in the environment.     

Cheater genotypes in the parthenogenetic ant Pristomyrmex punctatus

Cooperation is subject to cheating strategies that exploit the benefits of cooperation without paying the fair costs, and it has been a major goal of evolutionary biology to explain the origin and maintenance of cooperation against such cheaters. Here, we report that cheater genotypes indeed coexist in field colonies of a social insect, the parthenogenetic ant Pristomyrmex punctatus. The life history of this species is exceptional, in that there is no reproductive division of labour: all females fulfil both reproduction and cooperative tasks. Previous studies reported sporadic occurrence of larger individuals when compared with their nest-mates. These larger ants lay more eggs and hardly take part in cooperative tasks, resulting in lower fitness of the whole colony. Population genetic analysis showed that at least some of these large-bodied individuals form a genetically distinct lineage, isolated from cooperators by parthenogenesis. A phylogenetic study confirmed that this cheater lineage originated intraspecifically. Coexistence of cheaters and cooperators in this species provides a good model system to investigate the evolution of cooperation in nature.     

Degradation of wood and enzyme production by Ceriporiopsis subvermispora

The degradation of the components of Japanese beech and Japanese cedar wood was measured over time in cultures of the white-rot fungus Ceriporiopsis subvermispora. Although there was no initial degradation of cedar wood, after 12 weeks the mass loss of both cedar and beech wood was 15–20%. The mass losses of filter paper in beech wood-containing cultures and glucose cultures after 12 weeks were 87% and 70%, respectively. The ratio of lignin loss to mass loss of both beech and cedar wood cultures approached 2.0. Although the cellulose loss in cedar wood was very low throughout the 12-week incubation, C. subvermispora degraded the hemicellulose in Japanese cedar much more effectively than that in Japanese beech. These results confirm that C. subvermispora is a selective lignin degrader. During the 12-week incubation with Japanese beech wood, C. subvermispora continuously produced at least one of three phenol oxidases: laccase was produced initially, followed by Mn-independent peroxidase activity peaking at 6 weeks and Mn-dependent peroxidase activity peaking at 10 weeks. Lignin peroxidase and carboxymethylcellulase activities peaked after 3 weeks of incubation. Avicelase activity was present throughout the incubation period, although the activity was very low. The low-molecular-mass fraction of the extracellular medium, which catalyzes a redox reaction between O₂ and electron donors to produce hydroxyl radical, may act synergistically with the enzymes to degrade wood cell walls.     

Reproducibility and usability of chronic virus infection model using agent-based simulation; comparing with a mathematical model

We created agent-based models that visually simulate conditions of chronic viral infections using two software. The results from two models were consistent, when they have same parameters during the actual simulation. The simulation results comprise a transient phase and an equilibrium phase, and unlike the mathematical model, virus count transit smoothly to the equilibrium phase without overshooting which correlates with actual biology in vivo of certain viruses. We investigated the effects caused by varying all the parameters included in concept; increasing virus lifespan, uninfected cell lifespan, uninfected cell regeneration rate, virus production count from infected cells, and infection rate had positive effects to the virus count during the equilibrium period, whereas increasing the latent period, the lifespan-shortening ratio for infected cells, and the cell cycle speed had negative effects. Virus count at the start did not influence the equilibrium conditions, but it influenced the infection development rate. The space size had no intrinsic effect on the equilibrium period, but virus count maximized when the virus moving speed was twice the space size. These agent-based simulation models reproducibly provide a visual representation of the disease, and enable a simulation that encompasses parameters those are difficult to account for in a mathematical model.     

Establishment of variant PC12 subclones deficient in stimulation-secretion coupling

Clonal rat pheochromocytoma (PC12) cells have been widely used to study the molecular mechanism of exocytosis. We have isolated variant PC12 subclones with deficiencies in stimulation-secretion coupling, by a single cell recloning, and investigated the defects. PC12-1G2 hardly released dopamine following high-K(+)-induced depolarization, but normal release was evoked by the Ca(2+)-ionophore, ionomycin. Fura-2 fluorometry indicated that a nicardipine-sensitive component of Ca(2+) influx was missing, suggesting that PC12-1G2 has defects in L-type Ca(2+) channel function. PC12-2B3 was not responsive to high-K(+)-induced depolarization and ionomycin, and voltage-dependent Ca(2+) entry was identical to that of the normal clone. Electron microscopy revealed that the number of vesicles adjacent or directly attached to the plasma membrane was decreased in PC12-2B3. The expression of presynaptic proteins was analyzed by immunoblotting using a panel of antibodies. Syntaxin 1, VAMP-2, SNAP-25, Munc18, Rab3C and Sec-6 were decreased compared to the control clone and that of synaptophysin was extremely low. PC12-D60 synthesized and released dopamine normally, but had almost lost its catecholamine-uptake activity. These results show that multiple PC12 cells variants are spontaneously generated, and that recloning can select PC12 subclones useful for the study of the molecular mechanisms of neurotransmitter release.