G-quadruplex-forming aptamer enhances the peroxidase activity of myoglobin against luminol

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin''s peroxidase activity was discovered. Through our strategy—in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.     

Abrogation of CC chemokine receptor 9 ameliorates collagen-induced arthritis of mice

Introduction

Biological drugs are effective in patients with rheumatoid arthritis (RA), but increase severe infections. The CC chemokine receptor (CCR) 9 antagonist was effective for Crohn’s disease without critical adverse effects including infections in clinical trials. The present study was carried out to explore the pathogenic roles of chemokine (C-C motif) ligand (CCL) 25 and its receptor, CCR9, in autoimmune arthritis and to study if the CCR9 antagonist could be a new treatment for RA.

Methods

CCL25 and CCR9 expression was examined with immunohistochemistry and Western blotting. Concentration of interleukin (IL)-6, matrix metalloproteinase (MMP)-3 and tumor necrosis factor (TNF)-α was measured with enzyme-linked immunosorbent assays. Effects of abrogating CCR9 on collagen-induced arthritis (CIA) was evaluated using CCR9-deficient mice or the CCR9 antagonist, CCX8037. Fluorescence labeled-CD11b⁺ splenocytes from CIA mice were transferred to recipient CIA mice and those infiltrating into the synovial tissues of the recipient mice were counted.

Results

CCL25 and CCR9 proteins were found in the RA synovial tissues. CCR9 was expressed on macrophages, fibroblast-like synoviocytes (FLS) and dendritic cells in the synovial tissues. Stimulation with CCL25 increased IL-6 and MMP-3 production from RA FLS, and IL-6 and TNF-α production from peripheral blood monocytes. CIA was suppressed in CCR9-deficient mice. CCX8037 also inhibited CIA and the migration of transferred CD11b⁺ splenocytes into the synovial tissues.

Conclusions

The interaction between CCL25 and CCR9 may play important roles in cell infiltration into the RA synovial tissues and inflammatory mediator production. Blocking CCL25 or CCR9 may represent a novel safe therapy for RA.     

The Relationship between Media Consumption and Health-Related Anxieties after the Fukushima Daiichi Nuclear Disaster

Background

The Fukushima Daiichi nuclear disaster caused a global panic by a release of harmful radionuclides. In a disaster setting, misusage of contemporary media sources available today can lead to disseminated incorrect information and panic. The study aims to build a scale which examines associations between media and individual anxieties, and to propose effective media usages for future disaster management.

Methods

The University of Tokyo collaborated with the Fukushima local government to conduct a radiation-health-seminar for a total of 1560 residents, at 12 different locations in Fukushima. A 13 item questionnaire collected once before and after a radiation-seminar was used on factor analysis to develop sub-scales for multiple regression models, to determine relationships between the sub-scales and media type consumed. A paired t–test was used to examine any changes in sub-scale of pre- and post-seminar scores.

Results

Three sub-scales were revealed and were associated with different media types: was with rumors, while concern for the future was positively associated with regional-newspapers and negatively with national-newspapers. Anxiety about social-disruption was associated with radio. The seminar had a significant effect on anxiety reduction for all the three sub-scales.

Conclusion

Different media types were associated with various heightened concerns, and that a radiation seminar was helpful to reduce anxieties in the post-disaster setting. By tailoring post-disaster messages via specific media types, i.e., radio, it may be possible to effectively convey important information, as well as to calm fears about particular elements of post-disaster recovery and to combat rumors.     

Induction of GPR40 positively regulates cell motile and growth activities in breast cancer MCF-7 cells

Abstract

Free fatty acid (FFA) receptors belong to a member of G-protein-coupled receptors. GPCR 120 (GPR120) and GPR40 are identified as FFA receptors and activated via the binding of long- and medium-chain FFAs. The aim of this study was to assess the effects of GPR120 and GPR40 on cell motility and growth in breast cancer cells treated with tamoxifen (TAM). MCF-7 cells were continuously treated with TAM for approximately 6?months. The expression level of GPR40 gene was markedly higher in the long-term TAM treated (MCF-TAM) cells than in MCF-7 cells. In cell motility assay, MCF-TAM cells indicated the high cell motile activity, compared with MCF-7 cells. The cell motile activity of MCF-TAM cells was suppressed by a selective GPR40 antagonist, GW1100. To evaluate the effects of GPR40 on cell growth activity under estrogen-free conditions, cells were maintained in serum-free DMEM without phenol red for 2?days. In estrogen-free conditioned medium, the cell growth rate of MCF-TAM cells was significantly higher than that of MCF-7 cells. In addition, treatment of GW1100 reduced the cell growth rate of MCF-TAM cells. These results suggest that the cell motile and growth activities may be positively regulated through the induction of GPR40 by the long-term TAM treatment in MCF-7 cells.     

Acetyl-Ile-Gly-Leu protects neurons from Aβ1–42 induced toxicity in vitro and in V337M human tau-expressing mice

AimsWe previously reported that the neurotoxicity of amyloid β protein (Aβ_1–42, 10 nM) was blocked by an Aβ-derived tripeptide, Aβ_32–34 (Ile-Gly-Leu, IGL), suggesting that IGL may be a lead compound in the design of Aβ antagonists. In the present study, three stable forms of IGL peptide with acetylation of its N-terminal and/or amidation of its C-terminal (acetyl-IGL, IGL-NH₂ and acetyl-IGL-NH₂) were synthesized and examined for their effects on Aβ-induced neurotoxicity.Main methodsPhosphatidylinositol 4-kinase type II (PI4KII) activity was measured using recombinant human PI4KIIα kinase and cell viability was assessed in primary cultured hippocampal neurons. To test effects in vivo, 1.5 μl of 100 nM Aβ and/or 100 nM acetyl-IGL was injected into the hippocampal CA1 region of right hemisphere in transgenic mice expressing V337M human tau protein. Four weeks later, behavior performance in the Morris water maze was tested and after another 2 weeks, sections of brain were prepared for immunohistochemistry.Key findingsAmong the three modified tripeptides, acetyl-IGL attenuated the Aβ-induced inhibition of PI4KII activity as well as enhancement of glutamate neurotoxicity in primary cultured rat hippocampal neurons. Injection of Aβ into the hippocampus of mice impaired spatial memory and increased the number of degenerating neurons in bilateral hippocampal regions. Co-injection of acetyl-IGL prevented the learning impairment as well as the neuronal degeneration induced by Aβ.SignificanceThese results suggest that a modified tripeptide, acetyl-IGL, may be effective in the treatment of Alzheimer's disease.     

Ciliates expel environmental legionella-laden pellets to stockpile food

When Tetrahymena ciliates are cultured with Legionella pneumophila, the ciliates expel bacteria packaged in free spherical pellets. Why the ciliates expel these pellets remains unclear. Hence, we determined the optimal conditions for pellet expulsion and assessed whether pellet expulsion contributes to the maintenance of growth and the survival of ciliates. When incubated with environmental L. pneumophila, the ciliates expelled the pellets maximally at 2 days after infection. Heat-killed bacteria failed to produce pellets from ciliates, and there was no obvious difference in pellet production among the ciliates or bacterial strains. Morphological studies assessing lipid accumulation showed that pellets contained tightly packed bacteria with rapid lipid accumulation and were composed of the layers of membranes; bacterial culturability in the pellets rapidly decreased, in contrast to what was seen in ciliate-free culture, although the bacteria maintained membrane integrity in the pellets. Furthermore, ciliates newly cultured with pellets were maintained and grew vigorously compared with those without pellets. In contrast, a human L. pneumophila isolate killed ciliates 7 days postinfection in a Dot/Icm-dependent manner, and pellets harboring this strain did not support ciliate growth. Also, pellets harboring the human isolate were resuscitated by coculturing with amoebae, depending on Dot/Icm expression. Thus, while ciliates expel pellet-packaged environmental L. pneumophila for stockpiling food, the pellets packaging the human isolate are harmful to ciliate survival, which may be of clinical significance.     

Possibility of cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease mainly localizes on the mitochondria

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.     

Embryonic development of the sea butterfly Desmopterus papilio (Gastropoda: Thecosomata)

The embryonic development of the thecosome Desmopterus papilio is described for the first time. The mature individual produced a round-shaped egg mass containing ca. 200 fertilised eggs. First cleavage was observed 15 min after the release of the egg mass. Embryos showed typical molluscan spiral cleavage: macromeres produced the first and second quartets of micromeres in clockwise and counterclockwise directions, respectively. A trochophore larva hatched from the egg capsule 28 h after the release of the egg mass. Thereafter, the larva secreted a primary shell at the posterior part, developing into the veliger stage. These findings may be useful for future work on postembryonic development, especially on the loss of the veliger shell, in the genus Desmopterus which is the only group of thecosome species without a shell in the adult stage.