Development of a label-free immunosensor system for detecting plasma cortisol levels in fish

Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol levels in fish. In the present study, we describe a novel label-free immunosensor for detecting plasma cortisol levels. The method is based on immunologic reactions and amperometric measurement using cyclic voltammetry. For the immobilization of the antibody on the surface of sensing electrode, we used a self-assembled monolayer of thiol-containing compounds. Using this electrode, we detect the CV signal change caused by the generation of antigen–antibody complex. The immunosensor showed a response to cortisol levels, and the anodic peak value linearly decreased with a correlation coefficient of 0.990 in diluted plasma. The specificity of the label-free immunosensor system was investigated using other steroid hormones, such as 17α, 20β-dihydroxy-4-pregnen-3-one, progesterone, estriol, estradiol, and testosterone. The specific detection of cortisol was suggested by a minimal change from ?0.32 to 0.51 μA in the anodic peak value of the other steroid hormones. The sensor system was used to determine the plasma cortisol levels in Nile tilapia (Oreochromis niloticus), and the results were compared with those of the same samples determined using the conventional method (ELISA). A good correlation was obtained between values determined using both methods (correlation coefficient 0.993). These findings suggest that the proposed label-free immunosensor could be useful for rapid and convenient analysis of cortisol levels in fish plasma samples.     

Distribution and abundance of copepod nauplii in southern part of the East China Sea: implications for prey availability to jack mackerel Trachurus japonicus larvae

In the southern part of the East China Sea (ECS), a large spawning ground of jack mackerel Trachurus japonicus has recently been found: Larval survival during the period of transport from the spawning ground along the shelf break is potentially a critical stage prior to recruitment. As such, the distribution of copepod nauplii in this region was investigated during the main spawning period in 2003–2006. The average naupliar density in 2003 was significantly higher than the other years along the shelf break, which is a major transport pathway for jack mackerel larvae (sea-surface temperature 20–23°C). Estimated egg production rates for Paracalanus spp., one of the most dominant genera of copepods in the southern ECS, based on temperature and chlorophyll-a concentration could not fully explain the spatial and annual variation in naupliar distribution and abundance. Although naupliar densities showed significant positive correlations with chlorophyll-a concentration for all years, an analysis of covariance revealed that naupliar density in 2003 was high even if the effect of chlorophyll-a concentration was excluded. This suggests that apart from copepod production, adult female distribution plays an important role in variability of the naupliar distribution and abundance in the southern ECS.     

Effect of wind stress on the catch of Japanese anchovy Engraulis japonicus off northwestern Kyushu, Japan

Effect of wind stress on the annual catch of Japanese anchovy Engraulis japonicus off northwestern Kyushu for the period between 1963 and 2009 was investigated. Regime shift analysis detected several step changes in catch and environmental variables. Since the mid-1980s, the anchovy catch in the coastal fishery zones has declined, while the catch in the offshore zone has increased. The decline of catch in the coastal zones showed a significant correlation with the long-term variations in prevailing north-northeastward wind stress over the Goto-Nada Sea during spring spawning season. The results indicated that weakened north-northeastward winds caused the recent low recruitment of anchovy through low levels of wind-induced eggs and larval transport from the offshore spawning ground to the coastal nursery areas, resulting in the potential shift of nursery area to the northwestern offshore region. Thus, as well as the growth-favorable ambient temperature, transport process would play a key role on long-term fluctuations in anchovy abundance in these coastal seas.     

Cadmium detoxification effect of Chinese parsley Coriandrum sativum in liver and kidney of rainbow trout Oncorhynchus mykiss

Although fish feed supplemented with Chinese parsley (CP), Coriandrum sativum, does not affect the health of rainbow trout Oncorhynchus mykiss, it has the remarkable effect of reducing the uptake of Cd (12–17% compared with controls without CP). Here, we measured the change in the amount of metallothionein (MT) in the liver and kidney using gel permeation chromatography-high-performance liquid chromatography (TSK GEL SW 3000 column). The increase in MT content over time in the Cd-fed groups corresponded to the change in Cd accumulation. Gel permeation was performed with soluble fractions from the fish liver and kidney, and changes in the Cd, Cu, and Zn concentrations over time for each fraction, and the relationship between the form of Cd that accumulated in tissues and its toxicity were measured. A nontoxic MT fraction of 7,000 Da and a high-molecular-weight fraction of approximately 60,000 Da were isolated from the soluble fractions. The induction of nontoxic MT–Cd was higher in the CP-supplemented groups than in the control groups. The synergetic actions of several compounds in CP may contribute to bind MT, thereby preventing accumulation of harmful heavy metals and essential metals in fish.     

The Composition of Volatile Aroma Components, Flavanones, and Polymethoxylated Flavones in Shiikuwasha (Citrus depressa Hayata) Peels of Different Cultivation Lines

Citrus peels are important sources of various pleasant aroma compounds and valuable bioactive substances. To investigate differences in the composition and content of Shiikuwasha (Citrus depressa Hayata) peels from different cultivation lines, the composition of volatile aroma components, flavanones, and polymethoxylated flavones (PMFs) in four Shiikuwasha cultivation lines was examined. The composition of volatile aroma components in cold-pressed extracts of Shiikuwasha peels was analyzed using gas chromatography-flame ionization detection and gas chromatography-mass spectrophotometry. The extracts contained mainly monoterpene hydrocarbons (93.40-97.25%), including limonene (46.52-68.26%) and γ-terpinene (21.48-30.52%). Differences in the composition of volatile aroma compounds in the Shiikuwasha cultivation lines were revealed using principal component analysis. Additionally, the composition of flavanones and PMFs was determined using high-performance liquid chromatography methods. Neohesperidin (96.58%) was the predominant flavanone in 'Izumi kugani' peel, while the other peels had high hesperidin contents (89.26-98.66%). Moreover, the PMFs of Shiikuwasha peels were composed of nobiletin (56.74-64.77%) and tangeretin (23.17-34.70%).     

Nippon Dojohiryogaku Zasshi

The works (2,3,6, 7, 8) that have been reo ported so far on the study of application of carbonaceous materials with or without mineral nitrogen to soil indicate that these materials immediately tie up mineral nitrogen and its release occurs after a time. Broadbent et al. (2) said that if mineral nitrogen is once immobilized, its relese is very slow. Any conditions, therefore, which are concerned with the quantity of available nitrogen compounds and with the processes by which available nitrogen is produced or rendered unavailable are of great practical importance. Various circumstances surrounding these immobilization and release of nitrogen have long been subjects of research. But more evidences are needed before we draw sweeping conclusions for all conditions.     

Factors affecting immobilization and release of nitrogen in soil and chemical characteristics of the nitrogen newly immobilized III. Transformation of the nitrogen immobilized in soil and its chemical characteristics

The immobilization and release process of nitrogen and the chemical characteristics of newly immobilized-, subsequently released-, and residual-N were studied in soils receiving KNO₃ labelled with ¹⁵N together with glucose, straw, and cellulose.

Immobilization of nitrogen proceeded rapidly and reached its maximum at incubation periods of 3 days to 8 weeks or more, varying with the kind of carbon sources added. Following the maximum tie-up of nitrogen a rapid release began. After 20 weeks the immobilized nitrogen was released at a considerable rate for each carbon source.

At the period of maximum immobilization of nitrogen added to soil there was a clear difference in the percentage distribution of various forms of organic nitrogen between applied-N and native-N. The former was higher in amino acid N and unidentified N and lower in nonhydrolyzable N than the latter, and almost the same in hexosamine N and ammonium N as the latter.

With regard to all carbon sources the principal form of organic nitrogen contributing to mineralization was amino acid N and its susceptibility to mineralization was much higher in the applied-N than in the native-N, and further, the susceptibility of hexosamine N to mineralization was considerably lower than amino acid N.

In view of these results, it was presumed that a major origin of the amino acid N contributing to the mineralization process might be peptide complex substances such as mucopeptides and structural proteins, which originate from the microbial cell walls remaining in soil as a residual nitrogen of newly immobilized-N.     

Cell death process initiated by a combination of aluminium and iron in suspension-cultured tobacco cells (Nicotiana tabacum): Apoptosis-like cell death mediated by calcium and proteinase

Aluminium (AI) enhances ferrous ion (Fe²⁺) -mediated lipid per oxidation which is the primary factor leading to cell death in nutrient medium in tobacco cells. Under these conditions, cell death processes were examined. Cells which had been treated with AI and Fe²⁺ together exhibited cell shrinkage and DNA ladders which were detected by agarose-gel electrophoresis in the form of bands corresponding to increasing multiples of ～150 bp fragments. In the calcium (Ca)-free medium, a combination of Al and Fe2+ enhanced only partly the per oxidation of lipids and hardly promoted cell death, indicating that Ca²⁺ further stimulates the per oxidation of lipids enhanced by a combination of Al and Fe²⁺ and is required for cell death. N-Tosyl-L-phenylalanylchloromethyl ketone, an inhibitor of some cysteine and serine proteinases, had no effect on the per oxidation of lipids but almost completely blocked cell death enhanced by a combination of AI and Fe²⁺, suggesting that proteinase acts downstream from the per oxidation of lipids. On the basis of these findings, we conclude that cell death initiated by a combination of Al and Fe²⁺ is involved in the apoptosislike cell death program mediated by extracellular Ca²⁺ and endogenous proteinase.     

Responses to aluminium of suspension-cultured tobacco cells in a simple calcium solution

In a simple solution containing 3 mM CaCl₂ and 3% sucrose (pH 4.5), tobacco cells (Nicotiana tabacum L.) at the logarithmic phase of growth remained viable at least for 24 h. In this medium, the toxic effect of aluminium (Al) on the plasma membrane was investigated for up to 24 h. After the addition of Al to the cell suspension, Al started to accumulate immediately in the cells, and a maximum value was observed at 9 h. Al induced callose deposition, but did not enhance significantly the uptake of Evans blue (a nonpermeating dye), the per oxidation of lipids and the leakage of potassium (K) ions. Furthermore, the AI-treated cells were stained with fluorescein diacetate (FDA) as much as untreated control cells. These results suggest that the accumulation of Al does not damage the membrane. The addition of Fe(II) to the cells which had been exposed to Al for 12 h resulted in immediate lipid peroxidation and Evans blue uptake several hours later. A combination of Al and Fe(II) caused the K leakage, and enhanced the deposition of callose more than Al alone. These results suggest that the accumulation of Al sensitizes the membrane to the Fe(II)-mediated peroxidation of lipids, and that the Al-enhanced per oxidation of lipids is a direct cause of the loss of integrity of the plasma membrane (or cell death) in the Ca medium.     

Effects of soil type and nitrate concentration on denitrification products (N2O and N2) under flooded conditions in laboratory microcosms

Abstract

Denitrification products nitrous oxide ((N₂O) and nitrogen (N₂)) were measured in three flooded soils (paddy soil from Vietnam, PV; mangrove soil from Vietnam, MV; paddy soil from Japan, PJ) with different nitrate (NO₃^–) concentrations. Closed incubation experiments were conducted in 100-mL bottles for 7 d at 25°C. Each bottle contained 2 g of air-dried soil and 25 mL solution with NO₃^– (concentration 0, 5 or 10 mg N L^?1) with or without acetylene (C₂H₂). The N₂O + N₂ emissions were estimated by the C₂H₂ inhibition method. Results showed that N₂O + N₂ emissions for 7 d were positively correlated with those of NO₃^– removal from solution with C₂H₂ (R² = 0.9872), indicating that most removed NO₃^– was transformed to N₂O and N₂ by denitrification. In PJ soil, N₂O and N₂ emissions were increased significantly (P < 0.05) by the addition of greater NO₃^– concentrations. However, N₂O and N₂ emissions from PV and MV soils were increased by the addition of 0 to 5 mg N L^?1, but not by 5 to 10 mg N L^?1. At 10 mg N L^?1, N₂ emissions for 7 d were greater in PJ soil (pH 7.0) than in PV (pH 5.8) or MV (pH 4.3) soils, while N₂O emissions were higher in PV and MV soils than in PJ soil. In MV soil, N₂O was the main product throughout the experiment. In conclusion, NO₃^– concentration and soil pH affected N₂O and N₂ emissions from three flooded soils.