Evaluation of the PATHFAST Chemiluminescent Enzyme Immunoassay for Measuring Progesterone in Whole Blood and Serum of Mares

Evaluation of a new chemiluminescent enzyme immunoassay, the PATHFAST assay system (PATHFAST), for measurement of circulating progesterone in mares was performed. Five mares at the mid-luteal stage were administrated a single i.m. injection of prostaglandin F2α analog (PGF2α; cloprostenol 250 μg/ml), and then blood samples were collected from the jugular vein at 0, 15, 30 and 45 min, at one-hour intervals until 24 and at 48 hr via a catheter in the jugular vein. To monitor the physiological changes in circulating progesterone in mares after induced luteolysis, concentrations of progesterone in whole blood and serum samples were measured by PATHFAST. In addition, concentrations of progesterone in serum samples measured by PATHFAST were compared with those measured by radioimmunoassay (RIA) and enzyme immunoassay (EIA). Using PATHFAST, the serum concentrations of progesterone in mares correlated highly with those of whole blood samples (r=0.9672, n=88). The serum concentrations of progesterone as measured by PATHFAST correlated well with RIA (r=0.9654, n=88) and EIA (r=0.9323, n=112). An abrupt decline in circulating progesterone in whole blood samples was observed within 2 hr (50%), followed by a gradual decline until 48 hr later. The results for progesterone in whole blood samples correlated highly with those in serum samples, and the declining pattern paralleled that of the serum samples. These results demonstrated that PATHFAST is useful in the equine clinic as an accurate diagnostic tool for rapid assay of progesterone within 26 min, using unextracted whole blood.     

Resistance to anthracnose is decreased by tissue culture but increased with longer acclimation in the resistant strawberry cultivar

Inoculation of tissue-cultured plants of strawberry cultivar Ichigochukanbohon-Nou2gou (Nou-2) with the anthracnose pathogen (Glomerella cingulata) results in wilting and plant death, whereas inoculation of strawberry runners grown in greenhouses results in leaf spots, not wilting or death. When tissue-cultured Nou-2 plants were acclimated for 3, 9 or 15 days, plant resistance to anthracnose increased as the acclimation period increased, suggesting that the resistance of Nou-2 may be induced by external factors. To clarify the mechanisms of resistance, we used cDNA microarray analyses to compare gene expressions among tissue-cultured and acclimated plants of the resistant cultivar Nou-2 and the susceptible cultivar Tochiotome. As a result, we identified 18 cDNA clones that were upregulated during acclimation in Nou-2 but not in Tochiotome. In a real-time RT-PCR of the 18 clones, the expression levels of three were significantly higher in acclimated plants of Nou-2. Two of the three clones showed close homology to enzymes related to flavonoid biosynthesis, i.e., leucoanthocyanidin dioxygenase (LDOX, ANS) and UDP-glucosyltransferase, putative (3-GT). The clones spotted onto the cDNA microarrays were rechecked, and 23 nonredundant cDNA clones of 13 enzymes were estimated to be flavonoid biosynthetic enzymes. Most of these clones were upregulated by acclimation in Nou-2.     

Quantification of phosphatidic acid in foodstuffs using a thin-layer-chromatography-imaging technique

Apical application of lysophosphatidic acid (LPA), a growth-factor-like phospholipid, was shown to prevent or restore gastrointestinal (GI) disorders, such as diarrhea and stomach ulcer, in experimental animals. Because LPA is formed from phosphatidic acid (PA) by the activity of digestive phospholipase A(2), PA is a potential component for dietary treatment of such GI disorders. Here, we quantified PA contained in 38 foodstuffs and 3 herbs by a thin-layer-chromatography-imaging technique. Vegetables belonging to Brassicaceae, such as cabbage leaves (700 nmol/g of wet weight) and Japanese radish leaves (570 nmol/g), contained higher amounts of PA than other foodstuffs. Amounts of PA in fruits, cereals, and starchy root vegetables were below 300 nmol/g. Animal foodstuffs contained low amounts of PA (<60 nmol/g). Interestingly, leaves of Mallotus japonicas, a Japanese edible herb used for treatment of stomach ulcer, had the highest PA (1410 nmol/g) among those examined. The data shown here will be useful for the development of dietary treatment for a damaged GI tract.     

What is the optimal density of larval seeding in Acropora corals?

In situ larval seeding is a low-cost technique that is currently under development for the large-scale restoration of coral populations. One problem that still needs to be solved is the preparation of coral larvae for seeding, i.e., how many larvae are required to restore a certain area? In this study, we focused on the relationship between the numbers of larvae, settlers, and survivors for three?months post-settlement to determine the optimal larval seeding density. A comparison of three different larval densities (low, middle, and high) indicated that the number of settlers was proportional to the larval density, suggesting that settler density is determined by the number of larvae supplied. However, the survival rate of settlers on high-density plates was much lower than the corresponding rates on low- or middle-density plates during the first month after settlement. Moreover, most of the seeded corals had not survived on the low-density plates at three?months after settlement. Therefore, the middle larval density (i.e., 5000?larvae?m^?2) appears to be optimal for seeding on grid plates.     

Degradation of myofibrils in cultured yellowtail <Emphasis Type="Italic">Seriola quinqueradiata</Emphasis> burnt meat: effects of a myofibril-bound EDTA-sensitive protease

“Burnt meat” is a term used to describe the white (pale, grainy, exudative) muscle of yellowtail or tuna. It (with lightness parameter, L* ≥ 55) was observed after 2 h storage in the suffocate in air (SA) 29°C group and after 4 h storage in the spinal cord destruction (SCD) 29°C group. In the SA 17°C group, burnt meat was also observed after 4 h storage. In contrast, the meat in the SCD 17°C group was normal until after 12 h storage. The myosin heavy chain (MHC) was more degraded than the other myofibrillar proteins, and some protein bands increased in the burnt meat. The protease that leads to the degradation of MHC was investigated using myofibrils from the meat. EDTA completely suppressed the degradation, indicating that a myofibril-bound EDTA-sensitive protease (MBESP) may exist in yellowtail muscle and this caused the degradation of MHC. The optimum pH and temperature of MBESP in yellowtail were 5.0 and 50–60°C, respectively.     

Determinants of the daily rhythm of blood fluidity

Background  

Numerous processes in the living body exhibit daily rhythmicity. In this study, we characterized a daily rhythm of blood fluidity and identified its determinants.     

An efficient molecular technique for sexing tiger pufferfish (fugu) and the occurrence of sex reversal in a hatchery population

The tiger pufferfish (fugu) is one of the most important food fishes in East Asia. Since its testes are regarded as a delicacy, sex determination is economically relevant. Previous studies have identified a missense single-nucleotide polymorphism (SNP) in the Amhr2 (anti-Müllerian hormone receptor type II) gene as a strong candidate for a master sex-determining polymorphism. To distinguish genotypic sex efficiently, we developed a high-resolution melting (HRM) assay for this SNP site. By screening 396 fish from two independent crosses reared under controlled conditions, we observed perfect concordance between the SNP genotype and phenotypic sex. Thus, this method holds great potential for use in high-throughput sexing. When analyzing 293 progeny from a third cross reared under unknown conditions, we unexpectedly found that 25 % of phenotypic males exhibited female genotype. These results suggest that environmental factors such as rearing conditions could influence the sex-determination pathway in pufferfish. Alternatively, genetic modifiers might override the signals from Amhr2. This finding raises a concern regarding enhanced stock management of this species, because sex-reversed fish could compromise the sex ratio in subsequent generations. The HRM assay will also be useful for monitoring the degree of sex reversal before release.     

Effects of acidic fog and ozone on the growth and physiological functions of Fagus crenata saplings

The effects of simulated acid fog (SAF) and ozone (O₃) stress on the growth and physiology of beech (Fagus crenata) saplings were investigated. Three-year-old beech saplings were exposed to SAFs of pH 3 and pH 5 (control) during May 2007 to July 2008. In each SAF treatment group, half of the saplings were exposed to 60 ppb of O₃ during September 2007 to July 2008. In comparison to the control saplings, those from the pH 3 treatment had lower total plant biomasses, epicuticular wax amounts, Ca²⁺ concentrations in their leaves, and lower starch concentrations in their leaves and roots. The effect of O₃ was significant only for the starch concentration in the roots, but the O₃ exposure also negatively affected the growth and physiology of beech saplings. Results show that acid fog exerts various severe effects, and that both chronic acid fog and O₃ exposure suppressed the physiological functions of beech saplings.     

Maturation-associated changes in internal distribution and intra-ovarian microdistribution of tetrodotoxin in the pufferfish <Emphasis Type="Italic">Takifugu pardalis</Emphasis>

To clarify the maturation-associated accumulation profile of tetrodotoxin (TTX) in the female pufferfish Takifugu pardalis, specimens collected from Omura Bay, Nagasaki Prefecture, Japan, from November through December, 2014, were separated based on the histology of ovarian sections into four groups: yolk vesicle stage (Yv), yolk globule stage (Yg)-1, Yg-2, and Yg-3. TTX quantification in each tissue in each group by liquid chromatography–mass spectrometry revealed that the TTX concentration [mouse unit (MU)/g] in the ovary increased from Yv to Yg-1, and the TTX amount (MU/individual) in the ovary increased from Yg-1 through Yg-3. In Yv, TTX in the skin accounted for 86% of the total TTX in the body, whereas from Yg-1 through Yg-3, ovarian TTX accounted for 53–76% of the total. In female specimens collected from the same bay from February to May 2015, the TTX amount in the ovary markedly decreased after spawning, and the TTX amount in the liver increased. Immunohistochemical observation of the ovarian sections indicated that TTX localized mainly in the nucleus of immature oocytes, but then distributes to the yolk vesicles and yolk globules as maturation advances, and is partly transferred to the egg membrane as spawning approaches.