Oxysterols As Indices of Oxidative Stress in Man After Paraquat Ingestion

The aim of this study is to evaluate oxidative stress in man after paraquat ingestion by analyzing 7 &#102 - and 7 &#103 -hydroperoxycholest-5-en-3 &#103 -ol (7 &#102 - and 7 &#103 -OOH) as well as oxysterols, cholesterol oxidation products, as indices of lipid peroxidation. Lung, kidney, and liver were collected at autopsy from seven patients with paraquat poisoning and seven controls matched for age and sex. We identified for the first time 7-ketocholesterol (7-keto) and 7-hydroxycholesterol (7 &#102 -OH and 7 &#103 -OH) in human kidney by LC-MS. Next, we quantified 7 &#102 -OOH and 7 &#103 -OOH by HPLC with postcolumn chemiluminescence as well as oxysterols by HPLC-UV. Both 7 &#102 -OOH and 7 &#103 -OOH detected in lung and kidney from the controls were as low as the paraquat group. In contrast, we found both 7-keto and 7 &#103 -OH in lung and 7-keto in kidney from the paraquat group were significantly higher than from the controls. This is the first report on accumulated oxysterols in lung and kidney from human paraquat poisoning. It seems to reflect greater oxidative stress in the pathology of paraquat intoxication.     

A Novel Alveolate in Bivalves with Chemosynthetic Bacteria Inhabiting Deep‐Sea Methane Seeps

It has recently been unveiled that a wide variety of microbial eukaryotes (protists) occur in chemosynthetic ecosystems, such as hydrothermal vents and methane seeps. However, there is little knowledge regarding protists associated with endemic animals inhabiting these environments. In the present study, utilizing PCR techniques, we detected fragments of the small subunit ribosomal RNA gene (SSU rRNA gene) from a particular protist from gill tissues of a significant fraction of the vesicomyid clams Calyptogena soyoae and C. okutanii complex and of the mussel Bathymodiolus platifrons and B. japonicus, all of which harbor chemosynthetic endosymbiont bacteria and dominate methane seeps in Sagami Bay, Japan. Based on the phylogeny of SSU rRNA gene, the organism in question was shown to belong to Alveolata. It is noteworthy that this protist did not affiliate with any known alveolate group, although being deeply branched within the lineage of Syndiniales, for which the monophyly was constantly recovered, but not robustly supported. In addition, the protist detected using PCR followed by sequencing was localized within gill epithelial cells of B. platifrons with whole‐mount fluorescence in situ hybridization. This protist may be an endoparasite or an endocommensal of Calyptogena spp. and Bathymodiolus spp., and possibly have physiological and ecological impacts on these bivalves.     

An In Vitro ES Cell-Based Clock Recapitulation Assay Model Identifies CK2α as an Endogenous Clock Regulator

We previously reported emergence and disappearance of circadian molecular oscillations during differentiation of mouse embryonic stem (ES) cells and reprogramming of differentiated cells, respectively. Here we present a robust and stringent in vitro circadian clock formation assay that recapitulates in vivo circadian phenotypes. This assay system first confirmed that a mutant ES cell line lacking Casein Kinase I delta (CKIδ) induced ∼3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKIδ null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2α) homozygous mutant ES cell line developed significantly longer (about 2.5 hours) periods of circadian clock oscillations after in vitro or in vivo differentiation. Moreover, revertant ES cell lines in which mutagenic vector sequences were deleted showed nearly wild type periods after differentiation, indicating that the abnormal circadian period of the mutant ES cell line originated from the mutation in the CK2α gene. Since CK2α deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2α in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygous mutant ES cells, demonstrating that an ES cell-based in vitro assay is available for circadian genetic screening.     

Transformation of Lipid Bodies Related to Hydrocarbon Accumulation in a Green Alga,Botryococcus braunii (Race B)

The colonial microalga Botryococcus braunii accumulates large quantities of hydrocarbons mainly in the extracellular space; most other oleaginous microalgae store lipids in the cytoplasm. Botryococcus braunii is classified into three principal races (A, B, and L) based on the types of hydrocarbons. Race B has attracted the most attention as an alternative to petroleum by its higher hydrocarbon contents than the other races and its hydrocarbon components, botryococcenes and methylsqualenes, both can be readily converted into biofuels. We studied race B using fluorescence and electron microscopy, and clarify the stage when extracellular hydrocarbon accumulation occurs during the cell cycle, in a correlation with the behavior and structural changes of the lipid bodies and discussed development of the algal colony. New accumulation of lipids on the cell surface occurred after cell division in the basolateral region of daughter cells. While lipid bodies were observed throughout the cell cycle, their size and inclusions were dynamically changing. When cells began dividing, the lipid bodies increased in size and inclusions until the extracellular accumulation of lipids started. Most of the lipids disappeared from the cytoplasm concomitant with the extracellular accumulation, and then reformed. We therefore hypothesize that lipid bodies produced during the growth of B. braunii are related to lipid secretion. New lipids secreted at the cell surface formed layers of oil droplets, to a maximum depth of six layers, and fused to form flattened, continuous sheets. The sheets that combined a pair of daughter cells remained during successive cellular divisions and the colony increased in size with increasing number of cells.     

Phosphorus toxicity disrupts Rubisco activation and reactive oxygen species defence systems by phytic acid accumulation in leaves

Phosphorus (P) is an essential mineral nutrient for plants. Nevertheless, excessive P accumulation in leaf mesophyll cells causes necrotic symptoms in land plants; this phenomenon is termed P toxicity. However, the detailed mechanisms underlying P toxicity in plants have not yet been elucidated. This study aimed to investigate the molecular mechanism of P toxicity in rice. We found that under excessive inorganic P (Pi) application, Rubisco activation decreased and photosynthesis was inhibited, leading to lipid peroxidation. Although the defence systems against reactive oxygen species accumulation were activated under excessive Pi application conditions, the Cu/Zn-type superoxide dismutase activities were inhibited. A metabolic analysis revealed that excessive Pi application led to an increase in the cytosolic sugar phosphate concentration and the activation of phytic acid synthesis. These conditions induced mRNA expression of genes that are activated under metal-deficient conditions, although metals did accumulate. These results suggest that P toxicity is triggered by the attenuation of both photosynthesis and metal availability within cells mediated by phytic acid accumulation. Here, we discuss the whole phenomenon of P toxicity, beginning from the accumulation of Pi within cells to death in land plants.     

Possible contribution of Bradyrhizobium on nitrogen fixation in sweet potatoes

Aims

Sweet potato (Ipomoea batatas) is known for its ability to grow under nitrogen-limited conditions. To clarify the possible contribution of biological nitrogen fixation, we tried to isolate and identify diazotrophic bacteria from sweet potatoes.

Methods

By using cultivation technique, we isolated putative endophytes, which possess nifH genes, from surface-sterilized sweet potatoes. Their nitrogen-fixing abilities were demonstrated by the acetylene reduction assay in a semi-solid malate medium and sweet potato extracts. We also examined the colonization of an isolated strain (AT1) in sweet potatoes and their influence on growth and nitrogen fixation in plants as assessed by an acetylene reduction assay and ¹⁵N-isotope dilution technique.

Results

The isolates were identified as strains of Bradyrhizobium sp. AT1, Paenibacillus sp. AS2 and Pseudomonas sp. T16 based on their 16S rRNA gene sequences. They showed acetylene reduction activity (ARA) in the semi-solid malate medium. Among them, B. sp. AT1 showed ARA in sweet potato extracts under micro-aerobic conditions whereas both P. sp. AS2 and P. sp. T16 showed no ARA. The inoculation of B. sp. AT1 to the sweet potatoes resulted in increases in the fresh weights and detection of ARA in the inoculated plants. Moreover, the reduction of ¹⁵N atom % was observed in the inoculated plants compared to uninoculated controls.

Conclusions

B. sp. AT1 actively expresses nitrogenase activity in sweet potatoes and may contribute to the nitrogen nutrition of host plants.     

Hyperthermia inhibits homologous recombination repair and sensitizes cells to ionizing radiation in a time‐ and temperature‐dependent manner

Hyperthermia has long been known as a radio‐sensitizing agent that displays anti‐tumor effects, and has been developed as a therapeutic application. The mechanisms of hyperthermia‐induced radio‐sensitization are highly associated with inhibition of DNA repair. Our investigations aimed to show how hyperthermia inactivate homologous recombination repair in the process of sensitizing cells to ionizing radiation by using a series of DNA repair deficient Chinese Hamster cells. Significant differences in cellular toxicity attributable to hyperthermia at and above 42.5°C were observed. In wild‐type and non‐homologous end joining repair mutants, cells in late S phase showed double the amount heat‐induced radio‐sensitization effects of G1‐phase cells. Both radiation‐induced DNA double strand breaks and chromatin damage resulting from hyperthermia exposure was measured to be approximately two times higher in G2‐phase cells than G0/G1 cells. Additionally, G2‐phase cells took approximately two times as long to repair DNA damage over time than G0/G1‐phase cells. To supplement these findings, radiation‐induced Rad51 foci formations at DNA double strand break sites were observed to gradually dissociate in response to the temperature and time of hyperthermia exposure. Dissociated Rad51 proteins subsequently re‐formed foci at damage sites with time, and occurred in a trend also related to temperature and time of hyperthermia exposure. These findings suggest Rad51's dissociation and subsequent reformation at DNA double strand break sites in response to varying hyperthermia conditions plays an important role in hyperthermia‐induced radio‐sensitization. J. Cell. Physiol. 228: 1473–1481, 2013. © 2012 Wiley Periodicals, Inc.     

Acquisition of Contextual Discrimination Involves the Appearance of a RAS-GRF1/p38 Mitogen-activated Protein (MAP) Kinase-mediated Signaling Pathway That Promotes Long Term Potentiation (LTP)

RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.     

Morphological and genetic relationship of two closely‐related giant water bugs: Appasus japonicus Vuillefroy and Appasus major Esaki (Heteroptera: Belostomatidae)

The East Asian giant water bug species Appasus japonicus Vuillefroy and Appasus major Esaki are aquatic hemipteran insects whose ranges overlap, particularly in the Japanese Archipelago and on the Korean Peninsula. In rare cases, the two species co‐occur. Furthermore, they are very similar ecologically and also morphologically, making their identification extremely difficult, and the possibility of hybridization has also been suggested. In the present study, we re‐examined their taxonomic validity, and the characteristics useful for identifying them. To re‐examine the morphological traits useful for distinguishing these two species, 222 specimens of A. japonicus collected from Japan, Korea, and China, and 132 specimens of A. major from Japan and Korea, were examined. We also performed molecular phylogenetic analyses based on the mitochondrial DNA 16S rRNA and cytochrome oxidase subunit I (COI) regions and the nuclear DNA Histone 3 region. Although the two species are very similar ecologically and also morphologically, they showed significant genetic differentiation. Thus, there is likely some form of reproductive isolation acting between them. Major morphological characteristics overlap extensively between A. japonicus and A. major, and no particular trait was identified as being effective for differentiating these species. All the morphological characteristics examined overlapped between A. japonicus and A. major. However, a principal component analysis based on all of the morphological characteristics revealed that, despite the overlap between these species, it was possible to morphologically distinguish them. Therefore, a more accurate identification becomes possible using multiple characteristics rather than a single characteristic. The male genital paralobes, evaluated as the most useful morphological characteristic, was effective with 100% probability for the Japanese Appasus species. However, for the Asian (i.e. Korean) specimens, this characteristic was not useful. On the other hand, the results of molecular phylogenetic analyses based on the mitochondrial DNA 16S rRNA and COI regions and the nuclear DNA Histone 3 region clearly showed significant genetic differentiation between the two species. Notably, the results for the mitochondrial COI region strongly supported the independence of each monophyletic group (i.e. validity of each species). Therefore, DNA barcoding based on the mitochondrial DNA COI region is also considered useful for the identification of A. japonicus and A. major. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 615–643.     

Close Genetic Relationship between Legionella pneumophila Serogroup 1 Isolates from Sputum Specimens and Puddles on Roads,as Determined by Sequence-Based Typing

We investigated the prevalence of Legionella species isolated from puddles on asphalt roads. In addition, we carried out sequence-based typing (SBT) analysis on the genetic relationship between L. pneumophila serogroup 1 (SG 1) isolates from puddles and from stock strains previously obtained from sputum specimens and public baths. Sixty-nine water samples were collected from puddles on roads at 6 fixed locations. Legionella species were detected in 33 samples (47.8%) regardless of season. Among the 325 isolates from puddles, strains of L. pneumophila SG 1, a major causative agent of Legionnaires'' disease, were the most frequently isolated (n = 62, 19.1%). Sixty-two isolates of L. pneumophila SG 1 from puddles were classified into 36 sequence types (STs) by SBT. ST120 and ST48 were identified as major STs. Environmental ST120 strains from puddles were found for the first time in this study. Among the 14 STs of the clinical isolates (n = 19), 4 STs (n = 6, 31.6%), including ST120, were also detected in isolates from puddles on roads, and the sources of infection in these cases remained unclear. The lag-1 gene, a tentative marker for clinical isolates, was prevalent in puddle isolates (61.3%). Our findings suggest that puddles on asphalt roads serve as potential reservoirs for L. pneumophila in the environment.