Escherichia coli NifS-like proteins provide selenium in the pathway for the biosynthesis of selenophosphate

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. Kinetic characterization revealed the K(m) value for selenide approached levels that are toxic to the cell. Our previous demonstration that a Se(0)-generating system consisting of l-selenocysteine and the Azotobacter vinelandii NifS protein can replace selenide for selenophosphate biosynthesis in vitro suggested a mechanism whereby cells can overcome selenide toxicity. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, have been overexpressed and characterized. All three enzymes act on selenocysteine and cysteine to produce Se(0) and S(0), respectively. In the present study, we demonstrate the ability of each E. coli NifS-like protein to function as a selenium delivery protein for the in vitro biosynthesis of selenophosphate by E. coli wild-type SPS. Significantly, the SPS (C17S) mutant, which is inactive in the standard in vitro assay with selenide as substrate, was found to exhibit detectable activity in the presence of CsdB, CSD, or IscS and l-selenocysteine. Taken together the ability of the NifS-like proteins to generate a selenium substrate for SPS and the activation of the SPS (C17S) mutant suggest a selenium delivery function for the proteins in vivo.     

Microhabitat size distribution affects local difference in community structure: Metazoan communities in treeholes

I studied the effect of treehole (microhabitat) size distribution in local habitats on geographic difference in aquatic metazoan community structure by comparing differences between two sites on Iriomote Island, and between Iriomote Island (subtropical) and Tsushima Island (temperate), in southwestern Japan. In treeholes at each local site, the amount of litter, the species richness and total biomass of metazoa were positively correlated with treehole capacity. Between the two sites on Iriomote Island (Shirahama and Komi), the amount of litter, biomass and species number per treehole was greater at Komi where the mean and variance of treehole size were greater, while the dependencies of these parameters on treehole capacity were common to both sites. Total species number was larger at Komi (2 predators and 20 saprophages) than at Shirahama (1 predator and 19 saprophages). Most of the dominant taxa colonized larger treeholes with higher probabilities, although one taxa showed the opposite trend. Treeholes on Tsushima were smaller than those on Iriomote. The metazoan fauna in treeholes consisted of 15 saprophages on Tsushima, being less richer than that on Iriomote Island which had 2 predators and 21 saprophages. However, the dependencies of litter amount and biomass on treehole capacity did not differ significantly between the islands, although treeholes on Iriomote harbored a greater number of species per treehole than those on Tsushima. This study indicated that there are general correlates between community structure within individual treeholes (infracommunity structure) and treehole capacity (microhabitat size). Therefore, microhabitat-size distribution is potentially a significant constraint of local community structure, and its variation may contribute to the variation in local and regional species richness.     

Phylogeography of the Coastal Mosquito Aedes togoi across Climatic Zones: Testing an Anthropogenic Dispersal Hypothesis

The coastal mosquito Aedes togoi occurs more or less continuously from subarctic to subtropic zones along the coasts of the Japanese islands and the East Asian mainland. It occurs also in tropical Southeast Asia and the North American Pacific coast, and the populations there are thought to have been introduced from Japan by ship. To test this hypothesis, the genetic divergence among geographic populations of A. togoi was studied using one mitochondrial and three nuclear gene sequences. We detected 71 mitochondrial haplotypes forming four lineages, with high nucleotide diversity around temperate Japan and declining towards peripheral ranges. The major lineage (L1) comprised 57 haplotypes from temperate and subarctic zones in Japan and Southeast Asia including southern China and Taiwan. Two other lineages were found from subtropical islands (L3) and a subarctic area (L4) of Japan. The Canadian population showed one unique haplotype (L2) diverged from the other lineages. In the combined nuclear gene tree, individuals with mitochondrial L4 haplotypes diverged from those with the other mitochondrial haplotypes L1—L3; although individuals with L1—L3 haplotypes showed shallow divergences in the nuclear gene sequences, individuals from Southeast Asia and Canada each formed a monophyletic group. Overall, the genetic composition of the Southeast Asian populations was closely related to that of temperate Japanese populations, suggesting recent gene flow between these regions. The Canadian population might have originated from anthropogenic introduction from somewhere in Asia, but the possibility that it could have spread across the Beringian land bridge cannot be ruled out.     

A conserved α helix of Bcs1, a mitochondrial AAA chaperone,is required for the Respiratory Complex III maturation

Bcs1 is a transmembrane chaperone in the mitochondrial inner membrane, and is required for the mitochondrial Respiratory Chain Complex III assembly. It has been shown that the highly-conserved C-terminal region of Bcs1 including the AAA ATPase domain in the matrix side is essential for the chaperone function. Here we describe the importance of the N-terminal short segment located in the intermembrane space in the Bcs1 function. Among the N-terminal 44 amino acid residues of yeast Bcs1, the first 37 residues are dispensable whereas a hydrophobic amino acid in the residue 38 is essential for integration of Rieske Iron-sulfur Protein into the premature Complex III from the mitochondrial matrix. Substitution of the residue 38 by a hydrophilic amino acid residue affects conformation of Bcs1 and interactions with other proteins. The evolutionarily-conserved short α helix of Bcs1 in the intermembrane space is an essential element for the chaperone function.     

2-Haloacrylate Hydratase,a New Class of Flavoenzyme That Catalyzes the Addition of Water to the Substrate for Dehalogenation

Enzymes catalyzing the conversion of organohalogen compounds are useful in the chemical industry and environmental technology. Here we report the occurrence of a new reduced flavin adenine dinucleotide (FAD) (FADH₂)-dependent enzyme that catalyzes the removal of a halogen atom from an unsaturated aliphatic organohalogen compound by the addition of a water molecule to the substrate. A soil bacterium, Pseudomonas sp. strain YL, inducibly produced a protein named Caa67_YL when the cells were grown on 2-chloroacrylate (2-CAA). The caa67_YL gene encoded a protein of 547 amino acid residues (M_r of 59,301), which shared weak but significant sequence similarity with various flavoenzymes and contained a nucleotide-binding motif. We found that 2-CAA is converted into pyruvate when the reaction was carried out with purified Caa67_YL in the presence of FAD and a reducing agent [NAD(P)H or sodium dithionite] under anaerobic conditions. The reducing agent was not stoichiometrically consumed during this reaction, suggesting that FADH₂ is conserved by regeneration in the catalytic cycle. When the reaction was carried out in the presence of H₂¹⁸O, [¹⁸O]pyruvate was produced. These results indicate that Caa67_YL catalyzes the hydration of 2-CAA to form 2-chloro-2-hydroxypropionate, which is chemically unstable and probably spontaneously dechlorinated to form pyruvate. 2-Bromoacrylate, but not other 2-CAA analogs such as acrylate and methacrylate, served as the substrate of Caa67_YL. Thus, we named this new enzyme 2-haloacrylate hydratase. The enzyme is very unusual in that it requires the reduced form of FAD for hydration, which involves no net change in the redox state of the coenzyme or substrate.Dehalogenases catalyze the removal of halogen atoms from organohalogen compounds. These enzymes have been attracting a great deal of attention partly because of their possible applications to the chemical industry and environmental technology. Several dehalogenases have been discovered and characterized (6, 11, 14, 17, 22). Some of them act on unsaturated aliphatic organohalogen compounds in which a halogen atom is bound to an sp²-hybridized carbon atom. Examples include various corrinoid/iron-sulfur cluster-containing reductive dehalogenases (1, 7), cis- and trans-3-chloroacrylic acid dehalogenases (4, 19), and LinF (maleylacetate reductase), which acts on 2-chloromaleylacetate (5).In order to gain more insight into the enzymatic dehalogenation of unsaturated aliphatic organohalogen compounds, we searched for microorganisms that dissimilate 2-chloroacrylate (2-CAA) as a sole source of carbon and energy (8). 2-CAA is a bacterial metabolite of 2-chloroallyl alcohol, an intermediate or by-product in the industrial synthesis of herbicides (26). Rats treated orally with the herbicides sulfallate, diallate, and triallate excrete urinary 2-CAA (16). Various halogenated acrylic acids are produced by a red alga (27). We obtained three 2-CAA-utilizing bacteria as a result of screening (8). For one of these bacteria, Burkholderia sp. strain WS, we previously discovered a new NADPH-dependent enzyme, 2-haloacrylate reductase (12, 13). Although this enzyme does not directly remove a halogen atom from the substrate, it is supposed to participate in the metabolism of 2-CAA by catalyzing the conversion of 2-CAA into l-2-chloropropionate, which is subsequently dehalogenated by l-2-haloacid dehalogenase.Another bacterium that we obtained, Pseudomonas sp. strain YL, also dissimilates 2-CAA. However, the metabolic fate of 2-CAA in this bacterium remains unclear. In the present study, we analyzed proteins from 2-CAA- and lactate-grown cells of Pseudomonas sp. YL by two-dimensional polyacrylamide gel electrophoresis (PAGE) and identified a 2-CAA-inducible protein. We found that the protein catalyzes the dehalogenation of 2-CAA by the addition of a water molecule to the substrate, representing a new family of dehalogenases that act on unsaturated aliphatic organohalogen compounds. Remarkably, the enzyme requires reduced flavin adenine dinucleotide (FAD) (FADH₂) for its activity, although the reaction does not involve a net change in the redox state of the coenzyme or substrate. Here we describe the occurrence and characteristics of this unusual flavoenzyme.     

TPA-induced multinucleation of a mesenchymal stem cell-like clone is mediated primarily by karyokinesis without cytokinesis, although cell-cell fusion also occurs

The 5F9A cell, which is a mesenchymal stem cell-like clone established from rat bone marrow substrate adherent cells, can differentiate into adipocytes and osteoblasts in vitro under the appropriate conditions. Multinucleated cells could be also induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in 5F9A cells. This effect was mediated by protein kinase C. Possible mechanisms of multinucleation by TPA were hypothesized to be either karyokinesis without cytokinesis or cell-cell fusion. By observation using time-lapse phase-contrast microscopy, we determined that the multinucleated cells were generated mainly by karyokinesis without cytokinesis. Cell fusion was studied using time-lapse photography, and confocal laser scanning microscopy using two differentially labeled cells. These techniques demonstrated that multinucleated 5F9A cells could be produced by cell fusion, albeit at a low frequency. We conclude that multinucleated 5F9A cells are formed primarily by karyokinesis without cytokinesis, although some cells are also formed by cell-cell fusion.     

Synthesis and pharmacological profile of serofendic acids A and B

We present efficient syntheses of serofendic acids A and B (SA-A and SA-B), novel neuroprotective substances isolated from fetal calf serum. Biological and pharmacological evaluation showed that SA-A and SA-B have potent protective action against glutamate-induced neurotoxicity, but do not interact directly with glutamate receptors. A pharmacokinetic study showed that they have good oral bioavailability in rats. The results indicate that SA-A and SA-B are potential lead compounds for candidate drugs to treat various neurological disorders.     

The E3 ligase HACE1 is a critical chromosome 6q21 tumor suppressor involved in multiple cancers

Transformation and cancer growth are regulated by the coordinate actions of oncogenes and tumor suppressors. Here, we show that the novel E3 ubiquitin ligase HACE1 is frequently downregulated in human tumors and maps to a region of chromosome 6q21 implicated in multiple human cancers. Genetic inactivation of HACE1 in mice results in the development of spontaneous, late-onset cancer. A second hit from either environmental triggers or genetic heterozygosity of another tumor suppressor, p53, markedly increased tumor incidence in a Hace1-deficient background. Re-expression of HACE1 in human tumor cells directly abrogates in vitro and in vivo tumor growth, whereas downregulation of HACE1 via siRNA allows non-tumorigenic human cells to form tumors in vivo. Mechanistically, the tumor-suppressor function of HACE1 is dependent on its E3 ligase activity and HACE1 controls adhesion-dependent growth and cell cycle progression during cell stress through degradation of cyclin D1. Thus, HACE1 is a candidate chromosome 6q21 tumor-suppressor gene involved in multiple cancers.     

Phylogeny of the Geometridae and the evolution of winter moths inferred from a simultaneous analysis of mitochondrial and nuclear genes

Geometridae is one of the most diverse families within the Lepidoptera, comprising nine subfamilies. Winter moths, which have a unique life history, are found in three subfamilies. To examine the phylogeny of the Geometridae at the subfamily level and determine the evolutionary history of winter moths, we constructed phylogenetic trees for all nine geometrid subfamilies using two mitochondrial and two nuclear gene sequences. Specimens of all subfamilies were sampled from Japan. Simultaneous analyses of the combined data from all genes revealed that the Geometridae comprised two major clades: one with subfamilies Larentiinae and Sterrhinae, and the other with the remaining seven subfamilies. The second clade included the largest subfamily, Ennominae, and the subfamily Archiearinae, which is traditionally considered to be an ancestral lineage of the Geometridae. The Larentiinae+Sterrhinae clade contained one winter moth lineage, and the second major clade consisted of three winter moth lineages, including Alsophilinae, which contains winter moths exclusively. Using a Bayesian inference of divergence times, we estimated that geometrids began to diverge 54 Mya (62-48 Mya), whereas winter moth lineages differentiated from non-winter moth lineages 34-12 Mya, during the global cooling events in the Oligocene and the early Miocene. The adaptation to cool climates may have been a preadaptation that facilitated the winter moth life cycle.     

Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

Background

The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen.

Results

Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion.

Conclusions

The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0039-2) contains supplementary material, which is available to authorized users.