Antiradical Capacity and Induction of Apoptosis on HeLa Cells by a <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> Extract

Jamapa bean is a black Phaseolus vulgaris variety rich in condensed tannins, anthocyanins and flavonols with interesting biological activities. The objective of this work was to evaluate the antiradical capacity (ARC) of a Jamapa bean methanolic extract (BME) and some of the proanthocyanidin-rich fractions derived from it, using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The effect of the BME on some proteins involved in apoptosis on HeLa cells was also evaluated. A strong correlation between proanthocyanidin concentration in BME and antiradical capacity was found, suggesting that these compounds contribute significantly to antiradical activity. BME was a better radical scavenger than butylated hydroxytoluene (45.6 and 33.9% ARC at 400 μM, respectively). Two proanthocyanidin-rich fractions obtained after a preliminary separation of the BME using Toyopearl (TP4 and TP6) exhibited a higher antiradical activity than the parent extract. The treatment of HeLa cells with 35 μg BME/ml/24 h increased the expression of Bax and Caspase-3, pro-apoptotic proteins (6.13 and 1.2 times for Caspase-3 and Bax, respectively). The mechanism of action of some proteins involved in apoptosis was also evaluated, and the results suggest that black Jamapa bean could be an important source of polyphenolic compounds with potential biological use as antioxidant and anticancer agents.     

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV‐3) and possible latency of CyHV‐3 by temperature shift in the cells

A new cell line named CCF‐K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV‐3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV‐3‐infected CCF‐K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV‐3 was produced stably in CCF‐K104 cells over 30 viral passages. Our findings showed that CCF‐K104 is a useful cell line for isolation and productive replication of CyHV‐3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real‐time PCR showed that CyHV‐3 was present with low viral DNA loads, suggesting that CyHV‐3 may establish latent infection in CCF‐K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV‐3 genome arranged in a head‐to‐tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV‐3.     

Nonrandom distribution of chum salmon stocks in the Bering Sea and the North Pacific Ocean estimated using mitochondrial DNA microarray

More than 1,000 age-identified chum salmon Oncorhynchus keta collected at 23 stations in the Bering Sea and the North Pacific Ocean in June to July 2003 were used to estimate their origin of stocks using a DNA microarray developed for analyzing the mitochondrial (mt)DNA haplotypes. The observed haplotype distribution was nearly the same as that reported previously for fish collected in September 2002 and 2003 in the present surveyed areas. A conditional maximum-likelihood method for estimation of stock compositions indicated that the Japanese stocks mainly distributed in north central Bering Sea, whereas the Russian stocks were mainly in western Bering Sea. The North American stocks were abundant in eastern Bering Sea and around the Aleutian Islands. Such an area-specific stock composition was not significantly different between mature and immature fish. Thus, the combined results of 2 years suggest that the distribution of chum salmon is nonrandom in the surveyed areas in summer and autumn, and that fish of the same origin migrate together to the same area irrespective of age.     

Estimation of the spawning grounds of chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis> and spotted mackerel <Emphasis Type="Italic">Scomber australasicus</Emphasis> in the East China Sea based on catch statistics and biometric data

The spawning grounds of the chub mackerel (Scomber japonicus) and spotted mackerel (Scomber australasicus) in the East China Sea were estimated based on catch statistics of the Japanese large- and medium-type purse seine fishery from 1992 to 2006. Biometric data were obtained from specimens caught by purse seiners in the East China Sea from 1998 to 2006. Gonadosomatic index (GSI) at 50% sexual maturity of chub mackerel and spotted mackerel females was 2.5 and 2.6, respectively. Using this criterion for GSI, chub mackerel larger than 275 mm and spotted mackerel larger than 310 mm in fork length were considered to be mature. Mature chub mackerel was observed in the area of 15–22°C sea surface temperature (SST), and mature spotted mackerel was observed in the area of 17–25°C SST. The spawning period of chub mackerel ranged from February to June, and that of spotted mackerel ranged from February to May in the East China Sea. The spawning grounds were estimated from the distributions of catch per unit effort (CPUE) of spawners and SST. As a result, the spawning ground of chub mackerel was estimated to be in the central and southern part of the East China Sea and the area west of Kyushu in February, March, and April, and in the central part of the East China Sea, the area west of Kyushu and Tsushima Straight in May, and in Tsushima Straight and western part of the Sea of Japan in June. The spawning ground of spotted mackerel was estimated to be in the central and southern part of the East China Sea and southern coastal area of Kyushu in February, March, and April, and the central and southern part of the East China Sea and the area west of Kyushu in May.     

Growth stimulation of juvenile abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> by feeding with salmon growth hormone in sodium alginate gel

We examined the ability of salmon growth hormone (sGH) in sodium alginate (SA) gel to accelerate the somatic growth of juvenile abalone, Haliotis discus hannai, by feeding. After the feeding of sGH at 50 μg or 100 μg in 350 mg SA gel, immunoreactivity to sGH in body fluid was maximal after 12 h, and was still detectable at 24 h. No immunoreactivity was observed in the control group. Following the feeding of sGH at 0.5 mg or 5 mg/8 g of SA gel at 7- and 14-day intervals, there was a greater increase in shell length and body weight than in the control. In abalone, sGH can be transported from food into the circulatory system and subsequently improve somatic growth.     

Effect of prebiotic xylooligosaccharides on growth performances and digestive enzyme activities of allogynogenetic crucian carp (<Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>)

The effect of prebiotic xylooligosaccharides (XOS) on the growth performance and digestive enzyme activities of the allogynogenetic crucian carp, Carassius auratus gibelio, was investigated. XOS was added to fish basal semi-purified diets at three concentrations by dry feed weight: diet 1, 50 mg kg⁻¹; diet 2, 100 mg kg⁻¹; diet 3, 200 mg kg⁻¹, respectively. Twelve aquaria (n = 20) with three replicates for each treatment group (diets 1–3) and control treated without XOS were used. Weights of all collected carp from each aquarium were determined at the initial phase and at the end of the experiment, and the carp survival was also determined by counting the individuals in each aquarium. After 45 days, there were significant differences (P < 0.05) in the relative gain rate (RGR), and daily weight gain (DWG) of diets 1–3 were compared with the control. However, the survival rate was not affected (P > 0.05) by the dietary treatments. For enzymatic analysis, dissection produced a crude mixture of intestine and hepatopancreas of each segment to measure. The protease activity in the intestine and hepatopancreas content of fish in diet 2 (487.37 ± 20.58 U g⁻¹ and 20.52 ± 1.93 U g⁻¹) were significantly different (P < 0.05) from that in the control (428.13 ± 23.26 U g⁻¹ and 12.81 ± 1.52 U g⁻¹) and diet 3 (428.00 ± 23.78 U g⁻¹ and 14.04 ± 1.59 U g⁻¹). Amylase activity in the intestine was significantly higher for diet 2 compared to diet 1 and the control. As for amylase in the hepatopancreas, assays showed higher activity in diet 2 (P < 0.05) compared to the rest.     

Development of a whole-body cortisol extraction procedure for determination of stress in golden shiners, <Emphasis Type="Italic">Notemigonus crysoleucas</Emphasis>

Baitfish such as golden shiners are subjected to stress during harvesting, grading, and transport. Their small size makes it difficult to measure the stress response with the biological indicator cortisol using conventional assay methods for plasma. This paper examines the development and validation of methods for whole-body cortisol extraction from individual baitfish. Three types of extracts were tested: (1) an ethyl ether unaltered extract (UA); (2) an extract reconstituted in phosphate buffered saline (PBS); (3) an extract that had been increased in volume by the addition of food-grade vegetable oil (VO). These extracts were evaluated using validation tests with radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). The UA extract produced inadequate volumes of extract for multiple assays and could not be used for the determination of cortisol in a single fish. The PBS reconstitution method failed the precision recovery of serial dilutions (62.3%), linearity (R ²: 0.7864), and parallelism validation tests. The VO volume-boosting method passed all validation tests [intra-assay coefficent of variation (%CV): 16.3 for ELISA and 5.9 for RIA; inter-assay %CV: 10.3; spiked recovery: 102.0%; dilution recovery: 93.0%; linearity R ²: 0.9435; log of serial dilutions was parallel] and provided enough extract for multiple assays from an individual baitfish. Based on these results, we conclude that the VO volume-boosting method presents a means for determining cortisol from individual baitfish using either RIA or ELISA assays.     

Behavioural interactions of seabirds with suspended mussel longlines

Increased abundances of a number of seabird species have been found in areas of mussel longline aquaculture (compared to control sites) in a recent study at Bantry Bay, southwest Ireland. The aim of this study was to investigate whether this form of mussel aquaculture also affects the activity budgets and foraging behaviour of these seabirds. Scan-sampling was used to compare seabird activity budgets between three areas of mussel longline aquaculture and three control sites. Foraging activities of Great-Northern Divers Gavia immer, an Annex 1 species under the Euopean Union's Birds Directive, were also compared between sites using focal sampling. The behaviour of gulls (Larus sp.), Cormorants Phalacrocorax carbo, Shags Phalacrocorax aristotelis and Great-Northern Divers (but not auks, Alcidae) differed significantly between areas of mussel aquaculture and control sites in this study. Mussel suspension buoys were heavily utilized by gulls, cormorants and shags as safe perching platforms for preening activities. Gulls also fed extensively on epifauna attached to the buoys. Great-Northern Divers spent more time snorkelling (head submerged in search of food) in mussel sites than in the control sites, however dive duration and visible foraging success rates did not differ significantly between sites. Overall, the environmental impact of mussel suspension culture appears to be positive or neutral on marine bird species occurring at this study site.     

Influences of feeding frequency of extruded pellet and moist pellet on growth and body composition of juvenile Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> in suboptimal water temperatures

This study was carried out to investigate the influences of feeding frequency of extruded pellet and moist pellet on growth and body composition of juvenile flounder (initial mean weight 6.3 g) in sub optimal water temperatures. A 2 (diets: extruded pellet and moist pellet)×2 (feeding frequencies: two and three times daily)×2 (water temperatures: 12 and 17°C) factorial design with three replications was used. After 60 days of feeding, the feeding frequency did not significantly affect growth performance of fish. Weight gain, daily feed intake, feed efficiency and protein efficiency ratio were significantly (P<0.05) higher for fish reared at the higher water temperature. At the same water temperature, weight gain, feed efficiency, and protein efficiency ratio were significantly (P<0.05) higher for fish fed the extruded pellet than moist pellet. The results of this study indicate that feeding frequency of two times daily is sufficient for optimal growth of juvenile flounder when reared in suboptimal water temperatures, and an increase in water temperature from 12 to 17°C improved growth and feed efficiency.