Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells

Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F(2alpha) (PGF(2alpha)) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF(2alpha) concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF(2alpha) concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF(2alpha) concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.     

Synthesis and Structure–Activity Relationships of Miticidal 4,5-Dihydropyrazole-5-thiones

A series of novel 4,5-dihydropyrazole-5-thiones (DHPs) was synthesised by treating the corresponding dihydropyrazolones with ‘Lawesson’s reagent and evaluated for miticidal activity against two-spotted spider mites (Tetranychus urticae Koch). Of these, 3-(4-chlorophenyl)-4,4-dimethyl-1-phenyl-4,5-dihydropyrazole-5-thione, 3-(4-chlorophenyl)-4-ethyl-4-methyl-1-phenyl-4,5-dihydropyrazole-5-thione, 3-(4-chlorophenyl)-1-phenyl-4,5-dihydropyrazole-5-thione-4-spirocyclopentane and 4,4-dimethyl-1-phenyl-3-(4-trifluoromethyl-phenyl)-4,5-dihydropyrazole-5-thione were highly active (pEC₅₀>4·0) and were more effective than the miticide dicofol (pEC₅₀=3·879), which has traditionally been used for the control of phytophagous mites. Structure–activity relationship (SAR) studies were performed on each position of the pyrazole ring of DHPs. The results indicated that the unsubstituted phenyl, 4-substituted phenyl and thioxo groups on the 1-, 3- and 5-positions of DHPs respectively were required for activity. Quantitative SAR studies using physicochemical parameters of substituents and the capacity factor k′ as a hydrophobicity index suggested that: (a) the activities of all types of DHPs examined were mainly dominated by hydrophobicity, (b) the bulkiness of 4-substituents of the 3-phenyl ring favoured the activity and (c) the log k′ optimum for all DHPs was 1·675, equivalent to a log P_ow value of c. 5·0.     

The Effect of Methanolic Valeriana officinalis Root Extract on Adipocyte Differentiation and Adiponectin Production in 3T3-L1 Adipocytes

Plant Foods for Human Nutrition - Adipose tissue is an endocrine organ and its endocrine function is closely associated with type 2 diabetes mellitus. Valeriana officinalis (Valerian) exerts some...     

Identification of a resistant protein from scallop shell extract and its bile acid-binding activity

In this study, we showed that feeding rats the organic extract of scallop shells (scallop shell extract) caused a decrease in the weights of white adipose tissues in rats fed a high-fat diet. In addition, the cholesterol concentration in the serum of rats that received a diet containing scallop shell extract was significantly lower than that in the serum of rats on the control diet. Feeding this scallop shell extract to rats increased the fecal weight as well as the fecal excretion of bile acids. The amino acid composition of the feces from rats fed the scallop shell extract was different from that of feces from rats fed the control diet, and treatment of the extract with pepsin and pancreatin identified a protein with a molecular weight of 90 kDa (90-kDa protein) as one of the indigestible proteins. Interestingly the 90-kDa protein was found to be identical to a free radical-scavenging protein we previously identified and showed the ability to bind bile acids. These results suggest that indigestible proteins (resistant proteins) in the scallop shell extract, including the 90-kDa protein, inhibit the absorption of bile acid by binding to it and cause increased excretion of fecal bile acid, which subsequently may decrease the serum cholesterol level.     

Effect of artemia nauplii enriched with vitamin A palmitate on hypermelanosis on the blind side in juvenile Japanese flounder Paralichthys olivaceus

ABSTRACT:   The effect of Artemia nauplii enriched with different level of vitamin A (VA) palmitate (1 µg = 1 IU) on the occurrence of hypermelanosis on the blind side of Japanese flounder Paralichthys olivaceus was determined. Artemia were enriched with 0, 1, 2, 5 or 10 mg VA palmitate/L (control group, and 1-, 2-, 5-, and 10-mg groups). The enriched Artemia were fed to the larvae from 27 to 31 days post hatching (dph) corresponding to the F–G stage. VA palmitate, retinol and retinoic acid (RA) contents of Artemia were correlatively elevated with increasing VA palmitate in the culture medium. RA was detected in Artemia enriched with 5 mg and 10 mg, and a significantly high frequency of hypermelanosis on the blind side was observed in these groups at 65 dph ( P  < 0.05). These results suggest that RA synthesized from VA palmitate in Artemia could induce hypermelanosis on blind side of flounder when Artemia are enriched with more than 5 mg VA palmitate/L.     

Effects of high dose of vitamin A on reproduction and egg quality of Japanese flounder Paralichthys olivaceus

ABSTRACT: The present study was conducted to investigate the effects of dietary vitamin A on reproduction and egg quality in Japanese flounder Paralichthys olivaceus . Broodstock were fed experimental pellets containing two levels of vitamin A [11 × 10³ IU/100 g (control diet; CD), 337 × 10³ IU/100 g (experimental diet; ED)] for approximately 2 months before spawning and during the spawning period. Two groups of five females (average weight 1.4 kg) and 10 males (average weight 0.7 kg) were randomly allocated to two 30 m³ indoor tanks. Total egg production of the CD group was slightly higher than the ED group. Percentage of buoyant eggs and hatching rate of the ED group were significantly higher than the CD. In other egg quality parameters, such as percentage abnormal larvae and starvation tolerance of larvae, no notable difference was found between these two groups. At the end of the experiment, the skin color of broodstock in the ED group was darker than that of the CD group. Vitamin A content in eggs of the ED group was significantly higher than that of the CD group. However, the difference in vitamin A content in eggs between the ED and CD groups was much smaller than that in the liver of the females between the two groups. These results indicate that feeding broodstock a higher level of vitamin A increases the vitamin A content in eggs but does not affect egg quality in Japanese flounder because excess dietary vitamin A was stored mainly in the broodstocks' liver.     

Life-history variation in relation to nest site abundance in males of the freshwater goby Tridentiger brevispinis

Abstract –  Age at maturity in males of the freshwater goby Tridentiger brevispinis , a species with exclusive paternal care, was investigated in two populations in Lake Biwa, Japan, that differed markedly in nest site abundance. At Ohmi-maiko, where nest sites were scarce, most males matured at age 3, and mean body size of males guarding eggs in nests was larger than that of males sampled randomly (including both guarding and nonguarding males) in the population. Conversely, at Minamihama, where nest sites were abundant, many males matured at age 1, and there was no difference in body size between guarding males and males collected randomly. The slope of regression lines between body size of the guarding male and the number of eggs in his nest was greater in Ohmi-maiko than in Minamihama. These results suggest that the shortage of nest sites enhances reproductive success in larger males, probably through male–male competition for nest sites and female mate choice for larger males. I conclude that in nest spawners, the availability of nest sites should strongly affect life-history traits of males through sexual selection.     

Regulation of root-to-leaf Na and Cl transport and its association with photosynthetic activity in salt-tolerant soybean genotypes

ABSTRACT

Soil salinity is a major constraint to sustainable crop production. Genetic improvements are needed for growing soybean in salinity-prone environments. Salt-tolerant soybean genotypes alleviate a reduction in photosynthesis and growth under saline conditions; however, the detailed mechanisms involved remain unclear. Here, we aimed to clarify how Na and Cl root-to-leaf transport is quantitatively regulated, and to identify whether photosynthetic tolerance depends on traits associated with either stomata or with mesophyll tissues. Two pairs of pot-grown soybean near-isogenic lines (NILs) consisting of tolerant and susceptible counterparts, derived from a cross between salt-tolerant FT-Abyara and salt-sensitive C01, were subjected to salinity treatment in a rainout greenhouse. Comparison of photosynthetic responses between genotypes indicated that genotypic differences in salinity tolerance depended on the ability for sustained CO₂ assimilation in mesophyll tissues, rather than stomatal conductance. The ratio of photosynthetic rate to intercellular CO₂ concentration (A/Ci) declined exponentially with increasing Na and Cl concentration regardless of genotype, but tolerant genotypes effectively kept both elements at significantly low levels. Under saline conditions, tolerant genotypes reduced Na and Cl content at the two transport pathways: from root to stem, and from stem to leaf, but the reduction of Cl at each pathway was only minor. These results suggest that integrating genetic capacity for Cl transport regulation and osmotic adjustment should be an important target in salinity-tolerance soybean breeding.     

Improvement in recruitment of Japanese sardine with delays of the spring phytoplankton bloom in the Sea of Japan

To evaluate the impact of temporal variation of primary productivity on the recruitment of Japanese sardine (Sardinops melanostictus) in the Sea of Japan, the phenology of sea surface phytoplankton abundance was estimated from 8 day multiple satellite (SeaWiFS, MODIS‐Aqua, MERIS, and VIIRS) derived sea surface chlorophyll (SSChl) a concentrations from January 1998 to December 2015. Because relationships between SSChl a and in situ chlorophyll a concentrations were significantly different among periods based on the satellite combinations used, maximum and minimum SSChl a concentrations of 1 year were relativized as 1 and 0, respectively. Spatio‐temporal variation of relativized SSChl a concentrations was determined by using empirical orthogonal function (EOF) analysis. Scores in the first EOF mode denoted the basin‐scale variations of SSChl a concentrations in the Sea of Japan, and the major peak from the end of February to the end of May displayed the spring bloom. The logarithm of recruitment per spawner (LNRPS) for sardine was positively affected by delays in the start and end dates of the spring phytoplankton bloom. The delay of the date of the lowest sea surface temperature contributed to the delay of the end of the spring bloom during the period 1998–2015 and elevated the LNRPS during the period 1982–2015. Sardine spawns in the southern Sea of Japan from April to May, hence, delays of the spring bloom prolonged its overlap with sardine larval periods, and thus improved the recruitment of Japanese sardine in the Sea of Japan.     

Impact of land use on nitrogen concentration in groundwater and river water

Abstract

The aim of this study was to evaluate the impact of land use on nitrate nitrogen (NO₃-N) in shallow groundwater (G-N) and total nitrogen (N) in river water (R-N). The study area consisted of 26 watersheds (1342 km²) covering 72% of Kagawa Prefecture in Japan. We estimated G-N specific concentrations, which showed the magnitude of the upland fields, paddy fields, forests and urban land-use contributions to watershed-mean G-N. G-N specific concentrations were gained as partial regression coefficients using a multiple regression analysis of the watershed-mean G-N concentrations and the land-use ratios in each of the 26 watersheds. The results showed that the G-N specific concentration, which was gained as the partial regression coefficient for the multiple regression analysis, was 15.2 mg L^?1, 10.3 mg L^?1, 2.3 mg L^?1 and 2.5 mg L^?1 for the upland fields, paddy fields, forests and urban land-use types, respectively. R-N pollution load runoff to the river mouth was calculated by multiplying R-N specific concentration (previously reported) by river flow at the river mouth. Similarly, G-N pollution load arrival to groundwater was calculated by multiplying G-N specific concentration by the groundwater flow. The R-N pollution load runoff was 19.3 kg ha^?1 y^?1, 7.7 kg ha^?1 y^?1, 1.7 kg ha^?1 y^?1 and 7.6 kg ha^?1 y^?1, while the G-N pollution load arrival was 7.3 kg ha^?1 y^?1, 5.0 kg ha^?1 y^?1, 1.1 kg ha^?1 y^?1 and 1.2 kg ha^?1 y^?1, for upland fields, paddy fields, forests and urban areas, respectively. These results showed that the N in river water and groundwater was derived mainly from runoff and leaching from croplands. Therefore, the relationships between watershed-mean non-absorbed, applied nitrogen (NAA-N: nitrogen applied to cropland via fertilizer and manure without being absorbed by crops), R-N concentration and watershed-mean G-N concentration were investigated. A curvilinear correlation was observed between NAA-N and R-N concentrations (r² = 0.68) except for one small, high-density, urban watershed, and a weak linear correlation was observed between NAA-N and G-N concentrations (r² = 0.42).