A Bioluminescence Assay Using Nitrosomonas europaea for Rapid and Sensitive Detection of Nitrification Inhibitors

An expression vector for the luxAB genes, derived from Vibrio harveyi, was introduced into Nitrosomonas europaea. Although the recombinant strain produced bioluminescence due to the expression of the luxAB genes under normal growing conditions, the intensity of the light emission decreased immediately, in a time-and dose-dependent manner, with the addition of ammonia monooxygenase inhibitors, such as allylthiourea, phenol, and nitrapyrin. When whole cells were challenged with several nitrification inhibitors and toxic compounds, a close relationship was found between the change in the intensity of the light emission and the level of ammonia-oxidizing activity. The response of bioluminescence to the addition of allylthiourea was considerably faster than the change in the ammonia-oxidizing rate, measured as both the O₂ uptake and NO₂⁻ production rates. The bioluminescence of cells inactivated by ammonia monooxygenase inhibitor was recovered rapidly by the addition of certain substrates for hydroxylamine oxidoreductase. These results suggested that the inhibition of bioluminescence was caused by the immediate decrease of reducing power in the cell due to the inactivation of ammonia monooxygenase, as well as by the destruction of other cellular metabolic pathways. We conclude that the assay system using luminous Nitrosomonas can be applied as a rapid and sensitive detection test for nitrification inhibitors, and it will be used to monitor the nitrification process in wastewater treatment plants.     

Skeletal FGFR1 signaling is necessary for regulation of serum phosphate level by FGF23 and normal life span

Fibroblast growth factor (FGF) 23 produced by the bone is the principal hormone to regulate serum phosphate level. Serum FGF23 needs to be tightly regulated to maintain serum phosphate in a narrow range. Thus, we hypothesized that the bone has some phosphate-sensing mechanism to regulate the production of FGF23. Previously we showed that extracellular phosphate induces the phosphorylation of FGF receptor 1 (FGFR1) and FGFR1 signaling regulates the expression of Galnt3, whose product works to increase FGF23 production in vitro. In this study, we show the significance of FGFR1 in the regulated FGF23 production and serum phosphate level in vivo. We generated late-osteoblast/osteocyte-specific Fgfr1-knockout mice (Fgfr1^fl/fl; Ocn^Cre/+) by crossing the Ocn-Cre and the floxed Fgfr1 mouse lines. We evaluated serum phosphate and FGF23 levels, the expression of Galnt3 in the bone, the body weight and life span. A selective ablation of Fgfr1 aborted the increase of serum active full-length FGF23 and the enhanced expression of Galnt3 in the bone by a high phosphate diet. These mice showed more pronounced hyperphosphatemia compared with control mice. In addition, these mice fed with a control diet showed body weight loss after 23 weeks of age and shorter life span. These results reveal a novel significance of FGFR1 signaling in the phosphate metabolism and normal life span.     

Morphine-Induced Changes in Histamine Dynamics in Mouse Brain

The effect of the acute morphine treatment on histamine (HA) pools in the brain and the spinal cord was examined in mice. Morphine (1-50 mg/kg, s.c.) administered alone caused no significant change in the steady-state levels of HA and its major metabolite, tele-methylhistamine (t-MH), in the brain. However, depending on the doses tested, morphine significantly enhanced the pargyline (65 mg/kg, i.p.)-induced accumulation of t-MH and this effect was antagonized by naloxone. A specific inhibitor of histidine decarboxylase, alpha-fluoromethylhistidine (alpha-FMH) (50 mg/kg, i.p.), decreased the brain HA level in consequence of the almost complete depletion of the HA pool with a rapid turnover. Morphine further decreased the brain HA level in alpha-FMH-pretreated mice. Morphine administered alone significantly reduced the HA level in the spinal cord, an area where the turnover of HA is very slow. These results suggest that the acute morphine treatment increases the turnover of neuronal HA via opioid receptors, and this opiate also releases HA from a slowly turning over pool(s).     

Recessive Nonsense Suppressors in SACCHAROMYCES CEREVISIAE : Action Spectra, Complementation Groups and Map Positions

Three genes SUP111, SUP112 and SUP113 of Saccharomyces cerevisiae have been identified that can mutate to give recessive omnipotent nonsense suppressors. Alleles of these loci can also act as allosuppressors; that is, different phenotypes, due apparently to different efficiencies of suppression, can result from different alleles at a given locus. The SUP111, SUP112 and SUP113 loci map to the right arms of chromosomes VIII, VII and XIII, respectively.     

Hydrocarbon composition of newly isolated strains of the green microalga Botryococcus braunii

Four new strains of Botryococcus braunii were isolated from Japanese waters and cultured under defined conditions. Their hydrocarbon content and composition were analyzed and compared with those of the Darwin and Berkeley strains. The Yamanaka strain produced only alkadienes characteristic of the A race, whereas the others, the Yayoi, Kawaguchi-1 and -2 strains as well as the Darwin and Berkeley strains, produced botryococcenes peculiar to the B race. The hydrocarbon content of the Yamanaka strain was 16.1 % dry weight and that of the B race strains ranged from 9.7 to 37.9%. Botryococcene composition of the Japanese strains differed from each other as well as from the Darwin and Berkeley strains. More than 50% of the hydrocarbons in the Yayoi, Darwin, and Berkeley strains were composed of C₃₄H₅₈, but the main components were different from one another as isomers. The Kawaguchi-1 and -2 strains did not have a high level of C₃₄ botryococcenes, C₃₂ ones being the main components. In these strains significant amounts of squalene-related compounds were detected.     

Histological and Transcriptomic Analysis of Adult Japanese Medaka Sampled Onboard the International Space Station

To understand how humans adapt to the space environment, many experiments can be conducted on astronauts as they work aboard the Space Shuttle or the International Space Station (ISS). We also need animal experiments that can apply to human models and help prevent or solve the health issues we face in space travel. The Japanese medaka (Oryzias latipes) is a suitable model fish for studying space adaptation as evidenced by adults of the species having mated successfully in space during 15 days of flight during the second International Microgravity Laboratory mission in 1994. The eggs laid by the fish developed normally and hatched as juveniles in space. In 2012, another space experiment (“Medaka Osteoclast”) was conducted. Six-week-old male and female Japanese medaka (Cab strain osteoblast transgenic fish) were maintained in the Aquatic Habitat system for two months in the ISS. Fish of the same strain and age were used as the ground controls. Six fish were fixed with paraformaldehyde or kept in RNA stabilization reagent (n = 4) and dissected for tissue sampling after being returned to the ground, so that several principal investigators working on the project could share samples. Histology indicated no significant changes except in the ovary. However, the RNA-seq analysis of 5345 genes from six tissues revealed highly tissue-specific space responsiveness after a two-month stay in the ISS. Similar responsiveness was observed among the brain and eye, ovary and testis, and the liver and intestine. Among these six tissues, the intestine showed the highest space response with 10 genes categorized as oxidation–reduction processes (gene ontogeny term GO:0055114), and the expression levels of choriogenin precursor genes were suppressed in the ovary. Eleven genes including klf9, klf13, odc1, hsp70 and hif3a were upregulated in more than four of the tissues examined, thus suggesting common immunoregulatory and stress responses during space adaptation.     

Mate-searching behavior of the black chafer <Emphasis Type="Italic">Holotrichia kiotonensis</Emphasis> (Coleoptera: Scarabaeidae): identification of a sex pheromone,and male orientation behavior controlled by olfactory and visual cues

In the black chafer Holotrichia kiotonensis Brenske (Coleoptera: Scarabaeidae), mating behavior was observed between 1940 and 2010 hours at < 0.1 lx in both the laboratory and the field. In the laboratory, an ether extract of female abdominal glands induced a series of pre-mating behaviors such as short-distance orientation and abdominal bending. When the extract was fractionated by silica gel column chromatography, the active fraction was eluted with 50% ether in hexane and 100% ether. Gas chromatography–mass spectrometry analysis revealed that both active fractions contained anthranilic acid (2-amino-benzoic acid) as a major compound. The amount of this compound was determined to be ca. 600 ng/female by high performance liquid chromatography analysis with a fluorescence detector. In the field, male chafers were observed to land on cotton balls impregnated with 10 mg of authentic anthranilic acid. When a white ball treated with anthranilic acid was placed 2–10 cm away from an untreated black ball, males were observed to land significantly more frequently on the latter. These results suggest that males could recognize white balls below 0.1 lx and landed on black balls. When a treated black ball was placed beside an untreated black ball, more males landed on the former. The difference was significant when the distance between the two lures was 5 or 10 cm, but not significant when it was 2 cm. These observations demonstrated that anthranilic acid was the sex-attractant pheromone for the black chafer H. kiotonensis and that the males located and landed on a pheromone source by using olfaction in conjunction with visual orientation. The importance of visual orientation in this nocturnal species is discussed in comparison with the congeneric diurnal species Holotrichia loochooana loochooana.