Effect of Storage Temperature on Soil Nematode Community Structures as Revealed by PCR-DGGE

The optimal duration and conditions for storage of soils collected for nematode community analyses are unknown. To study this issue, three types of soils with different geographical origins from the subarctic to cool-temperate Japan were kept at three temperature levels (5, 10, and 20(°)C) for up to 8 wk following collection. During the storage period, nematode population density was measured, and community structure was assessed by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). No significant changes in the population density or diversity of nematodes (Shannon-Wiener Diversity Index) were observed during storage compared to initial states, except that density in an andosol collected from Tsukuba, Central Japan decreased significantly after 28 d of storage at 5(°)C. However, a regression analysis showed a declining trend in nematode density in the latter half of the storage period when soils were stored at 5 or 20(°)C, depending on the geographic origin of the soil. These results indicate that soils can be stored for 14 d at 5-20(°)C, with 10(°)C as optimal. This is the first study to experimentally determine the optimal preservation conditions for nematode assemblages in soils that are to be analyzed using PCR-DGGE.     

Ephexin4 and EphA2 mediate cell migration through a RhoG-dependent mechanism

EphA2, a member of the Eph receptor family, is frequently overexpressed in a variety of human cancers, including breast cancers, and promotes cancer cell motility and invasion independently of its ligand ephrin stimulation. In this study, we identify Ephexin4 as a guanine nucleotide exchange factor (GEF) for RhoG that interacts with EphA2 in breast cancer cells, and knockdown and rescue experiments show that Ephexin4 acts downstream of EphA2 to promote ligand-independent breast cancer cell migration and invasion toward epidermal growth factor through activation of RhoG. The activation of RhoG recruits its effector ELMO2 and a Rac GEF Dock4 to form a complex with EphA2 at the tips of cortactin-rich protrusions in migrating breast cancer cells. In addition, the Dock4-mediated Rac activation is required for breast cancer cell migration. Our findings reveal a novel link between EphA2 and Rac activation that contributes to the cell motility and invasiveness of breast cancer cells.     

Serine Residues in the Cytosolic Tail of the T-cell Antigen Receptor α-Chain Mediate Ubiquitination and Endoplasmic Reticulum-associated Degradation of the Unassembled Protein

The T-cell antigen receptor (TCR) α-chain (TCRα) is a type I integral membrane protein that becomes ubiquitinated and targeted to the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway when it fails to assemble into the heteromeric TCR complex. Remarkably, TCRα has a cytosolic tail of only five amino acid residues (i.e. RLWSS), none of which is the conventional ubiquitin acceptor, lysine. Herein we report that substitution of two conserved serine residues in the cytosolic tail of TCRα to alanine decreased ubiquitination, whereas placement of additional serine residues enhanced it. Moreover, replacement of the cytosolic serine residues by other ubiquitinatable residues (i.e. cysteine, threonine, or lysine) allowed ubiquitination to take place. Serine-dependent ubiquitination perfectly correlated with targeting of TCRα for ERAD. We also found that this ubiquitination was mediated by the ER-localized ubiquitin ligase, HRD1. These findings indicate that serine-dependent, HRD1-mediated ubiquitination targets TCRα to the ERAD pathway.     

Characterization of arylsulfatase formed by derepressed synthesis in Citrobacter braakii

Arylsulfatase activity was detected in a bacterial strain, Citrobacter braakii 69-b, isolated from soil by enrichment cultivation using porcine gastric mucin. The production of arylsulfatase was derepressed markedly in a synthetic medium by the addition of tyramine. The purified enzyme hydrolyzed 4-nitrophenyl sulfate, 4-nitrocatechol sulfate, and 3-indoxyl sulfate, and was classified as type I arylsulfatase.     

Chromosome-specific satellite sequences in Turritis glabra

Two novel repetitive sequence families were isolated from Turritis glabra (2n = 2x = 12). These two repeat families are similar to those of centromeric repeats in Arabidopsis thaliana, are co-localized on one chromosome pair, and differ by about 20% from each other. Phylogenetic analysis revealed that the two repeat families of T. glabra are more similar to each other than to the centromeric repeat families of other Arabidopsis and related species. The relationships of satellite sequences reflected the species phylogeny, indicating that the replacement of satellite sequences has occurred in each species lineage independently, and shared variants could not have existed for a long time between species.     

Effect of tea catechins on mitochondrial DNA 4977-bp deletions in human leucocytes

The catechins in green tea have antioxidative and antimutagenic effects. We examined the effect of green tea enriched with catechins on the presence of mitochondrial DNA (mtDNA) with a common 4977-bp deletion mutation (mtDNA4977) in human leucocytes. Ten healthy females [aged 20.80 +/- 1.03 years] drank 350 ml of catechin-rich tea daily after supper for 5 weeks. Blood samples were collected twice before, and twice after 5 weeks of consuming the tea. Deletions in mtDNA were analyzed using the nested polymerase chain reaction (PCR). We identified a common mtDNA4977 deletion in nine participants before drinking the tea. However, this mtDNA4977 deletion was not evident in leucocytes from most of the participants 5 weeks after drinking the tea. Catechins found in tea might contribute to the maintenance of health status by reducing damage to mtDNA and by maintaining the capacity of mtDNA for oxidative phosphorylation.     

Expression and distribution of Gpr119 in the pancreatic islets of mice and rats: predominant localization in pancreatic polypeptide-secreting PP-cells

The GPR119 was recently shown to be activated by oleoylethanolamide (OEA), a naturally occurring bioactive lipid with hypophagic and anti-obesity effects. In this study, we have cloned and characterized its murine counterpart, Gpr119. The full-length cDNA contained an open reading frame of 1008bp encoding a 335-amino acid protein. The genomic organization of Gpr119 was unique, having a 3'-untranslated second exon that was also involved in an alternative splicing event. Gene expression analyses confirmed its specific expressions in pancreatic islets and two endocrine cell-lines, MIN6 and alphaTC1. Immunohistochemistry and double-immunofluorescence studies using a specific antibody revealed the predominant Gpr119 localization in pancreatic polypeptide (PP)-cells of islets. No definitive evidence of Gpr119-immunoreactivity in adult beta- or alpha-cells was obtained. The Gpr119 mRNA levels were elevated in islets of obese hyperglycemic db/db mice as compared to control islets, suggesting a possible involvement of this receptor in the development of obesity and diabetes.     

Improving effect of ethyl eicosapentanoate on statin-induced rhabdomyolysis in Eisai hyperbilirubinemic rats

The effect of ethyl eicosapentanoate (EPA-E) on statin-induced rhabdomyolysis was investigated by co-administration of EPA-E and pravastatin (PV), as a typical statin, to Eisai hyperbilirubinemic rats (EHBR). It was confirmed that the plasma PV concentration was not affected by simultaneous administration of EPA-E, and there was no cumulative increase of PV during prolonged co-administration of EPA-E and PV. Muscular degeneration was prominent (incidence 5/5; average grade 3.5 (range 2-4)) in EHBR treated with PV alone at 200 mg/kg/day for 14 days, but co-administration of EPA-E at doses of 100, 300, and 1000 mg/kg/day decreased the average grades to 1.4 (range 0.3-3.0), 0.5 (0.2-1.0), and 0.6 (0.0-1.7), respectively. Creatine phosphokinase (CPK) and myoglobin levels in plasma were well correlated with the grade of skeletal muscle degeneration. Thus, EPA-E appears to reduce the severity of statin-induced rhabdomyolysis.     

Crohn's disease risk alleles on the NOD2 locus have been maintained by natural selection on standing variation

Risk alleles for complex diseases are widely spread throughout human populations. However, little is known about the geographic distribution and frequencies of risk alleles, which may contribute to differences in disease susceptibility and prevalence among populations. Here, we focus on Crohn's disease (CD) as a model for the evolutionary study of complex disease alleles. Recent genome-wide association studies and classical linkage analyses have identified more than 70 susceptible genomic regions for CD in Europeans, but only a few have been confirmed in non-European populations. Our analysis of eight European-specific susceptibility genes using HapMap data shows that at the NOD2 locus the CD-risk alleles are linked with a haplotype specific to CEU at a frequency that is significantly higher compared with the entire genome. We subsequently examined nine global populations and found that the CD-risk alleles spread through hitchhiking with a high-frequency haplotype (H1) exclusive to Europeans. To examine the neutrality of NOD2, we performed phylogenetic network analyses, coalescent simulation, protein structural prediction, characterization of mutation patterns, and estimations of population growth and time to most recent common ancestor (TMRCA). We found that while H1 was significantly prevalent in European populations, the H1 TMRCA predated human migration out of Africa. H1 is likely to have undergone negative selection because 1) the root of H1 genealogy is defined by a preexisting amino acid substitution that causes serious conformational changes to the NOD2 protein, 2) the haplotype has almost become extinct in Africa, and 3) the haplotype has not been affected by the recent European expansion reflected in the other haplotypes. Nevertheless, H1 has survived in European populations, suggesting that the haplotype is advantageous to this group. We propose that several CD-risk alleles, which destabilize and disrupt the NOD2 protein, have been maintained by natural selection on standing variation because the deleterious haplotype of NOD2 is advantageous in diploid individuals due to heterozygote advantage and/or intergenic interactions.