Somaclonal variation in Tricyrtis hirta plants regenerated from 1-year-old embryogenic callus cultures

The Liliaceous perennial Tricyrtis hirta, sometimes called ‘Japanese toad lily’, has recently become popular as an ornamental for pot and garden uses. Highly embryogenic callus cultures of this plant predominately consisted of diploid cells but also contained tetraploid cells after 1 year of establishment. In the present study, plans regenerated from the 1-year-old embryogenic callus cultures were subjected to ploidy level analysis and morphological characterization following 3 years of cultivation. Among 37 plants examined, 28 kept the diploid level (2n = 2x = 26) but nine were tetraploid (2n = 4x = 52) as indicated by FCM analysis and chromosome observation. Although no morphological alterations were detected in 26 out of 28 diploid regenerants, the remaining two showed noticeable variations: both were severely dwarf and had crimped leaves and many malformed flowers. The tetraploid regenerants had several horticulturally attractive characteristics compared with the diploid controls, such as longer shoots, thicker stems, and larger flowers. Thus regeneration of tetraploid plants from 1-year-old embryogenic callus cultures offers a possibility to improve the horticultural value of T. hirta, although regeneration of trueness-to-type plants is essential for utilizing the cultures for micropropagation and genetic transformation.     

Effects of TAK-044, a nonselective endothelin receptor antagonist, on the spontaneous and indomethacin- or methylene blue-induced constriction of the ductus arteriosus in rats

We studied the effects of TAK-044, a nonselective endothelin (ET) receptor antagonist, on the indomethacin- or methylene blue-induced constriction of the ductus arteriosus (DA) in rats and compared them with the effects on spontaneous DA constriction. Injection of TAK-044 into 21-day-old fetuses in utero was performed through the uterine wall of laparotomized mother rats under light ether anesthesia. The fetuses were autopsied 3 hr after treatment with TAK-044 (10 mg/kg) in utero and simultaneous administration to the laparotomized mother rats of indomethacin (3 mg/kg, p.o.) or methylene blue (100 mg/kg, i.p.). In the second experiment, pregnant rats were decapitated on day 21 of gestation to obtain newborn rats by cesarean delivery. Newborn rats which were given TAK-044 (2, 10 mg/kg) immediately after or 1 hr before cesarean delivery were autopsied at various times after birth. In both experiments, pups were rapidly frozen in an acetone-dry ice mixture at autopsy to evaluate the DA constriction by the whole-body freezing and shaving method. TAK-044 injection into the fetus 3 hr before autopsy completely inhibited the DA constriction induced by maternal treatment with indomethacin or methylene blue. TAK-044 caused dose-dependent inhibition of the spontaneous closure of the DA after birth. The inhibitory effect was more pronounced in pups which were given TAK-044 in utero 1 hr before birth; however, the inhibitory effect was incomplete in newborn pups. These results, together with the previous finding that BQ-123, an ETA-specific receptor antagonist, inhibits the ductal constriction induced by oxygen in vitro [Coceani et al., 1992], indicate that the ETA receptor plays a significant role in the indomethacin- or methylene blue-induced DA constriction as well as in the spontaneous DA constriction after birth, and also indicate that the inhibition of ETA receptor by TAK-044 was more easily achieved in fetuses than in neonates.     

Effects of Dietary Contamination by Zearalenone and Its Metabolites on Serum Anti‐Müllerian Hormone: Impact on the Reproductive Performance of Breeding Cows

We investigated the effects of in vivo exposure to low zearalenone levels on the anti‐Müllerian hormone endocrine levels and the reproductive performance of cattle. Urine and blood samples and reproductive records were collected from two Japanese Black breeding female cattle herds with dietary zearalenone contamination below the threshold levels (<1 ppm) at 30 days after calving. Urinary zearalenone, α‐zearalenol and β‐zearalenol concentrations were measured by chromatography‐tandem mass spectrometry, and serum anti‐Müllerian hormone concentrations were determined along with serum biochemical parameters. Urinary concentrations of α‐zearalenol were significantly higher (p < 0.05) in cattle in Herd 1 than in cattle in Herd 2, reflecting the different amounts of zearalenone in the diet of the two herds. Although the number of 5‐mm and 10‐mm follicles of the herds and their fertility after artificial insemination were similar, the serum anti‐Müllerian hormone concentrations in herds 1 and 2 were 438.9 ± 48.6 pg/ml and 618.9 ± 80.0 pg/ml, respectively, with a trend towards a significant difference (p = 0.053), which may indicate differences in the antral follicle populations between herds. Thus, zearalenone intake from dietary feed, even when below the threshold zearalenone contamination level permitted in Japan, may affect the ovarian antral follicle populations, but not the fertility, of post‐partum cows.     

Distribution of three algicidal <Emphasis Type="Italic">Alteromonas</Emphasis> sp. strains in seagrass beds and surrounding areas in the Seto Inland Sea,Japan

Harmful algal blooms (HABs) often cause great damage to the fish aquaculture industry in the western part of Japan. Bacteria that effectively kill such phytoplankton are found in coastal seawater and are considered to influence the occurrence of HABs. Algicidal bacteria are found in abundance in seagrass beds; however, the distribution of particular bacterial strains has not yet been clarified. In this study, we determined the abundance of three algicidal Alteromonas sp. strains that were isolated as Chattonella antiqua-killing bacteria in seagrass beds and their surrounding areas in summers of 2013–2015. The strains were detected using a quantitative polymerase chain reaction (qPCR) amplification method. Two of the three algicidal bacterial strains were remarkably abundant in the seawater of an enclosed seagrass bed compared to the surrounding areas. In addition, the abundance of the three algicidal bacteria decreased when the eelgrass withered in late summer. These results suggest that growth of these algicidal bacteria was stimulated by eelgrass. The bacterial abundance estimated by qPCR was much greater than that determined by the culture-dependent method in June 2015 when the eelgrass grew thickly. This implies that the qPCR assay could be a sensitive tool to evaluate algicidal bacteria in natural environments.     

Role of taurine deficiency in inducing green liver symptom and effect of dietary taurine supplementation in improving growth in juvenile red sea bream <Emphasis Type="Italic">Pagrus major</Emphasis> fed non-fishmeal diets based on soy protein concentrate

This study was conducted to investigate the mechanism of green liver symptom induction and the effect of dietary taurine supplementation on growth performance in juvenile red sea bream fed non-fishmeal diets based on soy protein concentrate (SPC). Juvenile fish (initial BW 72 g) were fed for 20 weeks on SPC diets supplemented with taurine at levels of 0, 1.0, and 2.0%. In the taurine-unsupplemented SPC diet group, specific growth rate (SGR) and feed conversion ratio (FCR) were significantly inferior (P < 0.001), and incidence of green liver was observed in 70% of fish. In this group, hepatopancreatic and plasma taurine concentrations were lowest (P < 0.05), hepatopancreatic content of bile pigments was highest (P < 0.05), and osmotic tolerance of erythrocytes was inferior (P < 0.05) among the dietary treatment groups. Serum osmolality of all treatment groups was at similar levels. These physiological abnormalities as well as SGR and FCR were improved by dietary taurine supplementation. These results indicate that the mechanism for induction of green liver symptom is bile pigment overproduction due to increased hemolysis because erythrocytes become osmotically fragile due to dietary taurine deficiency. Taurine supplementation of SPC diets is essential for maintaining normal physiological condition and growth performance in juvenile red sea bream.     

Biodegradation of chlorothalonil in soil after suppression of degradation

the degradation rate of chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) was significantly decreased after repeated application in field experiments. However, the degradation rate completely recovered after further application of chlorothalonil. Chlorothalonil was also degraded in a laboratory study, forming a stoichiometric amount of chloride anion. 4-Hydroxy-2,5,6-trichloroisophthalonitrile (TPN-OH) was also detected as a metabolite in the study. Bacterial which degrade chlorothalonil rapidly were isolated from the soil. The cell suspension of the bacteria transformed chlorothalonil to TPN-OH and chloride anion, but did not utilize chlorothalonil (=cometabolism). It seems that microbial acclimation has occurred during repeated application because the bacteria could not be detected in soil which was not treated with chlorothalonil.Dedicated to Professor J. C. G. Ottow on the occasion of his 60th birthday     

Time-course changes in 5-fluorouracil-induced neural progenitor cell damages in the developing rat brain

5-Fluorouracil (5-Fu) is a DNA-damaging agent and teratogenic in rodents. This study aimed to investigate its influence on neural progenitor cells (NPCs) in the developing fetal rat brain. Dams were intraperitoneally injected with 5-Fu (50 mg/kg b.w.) on gestation day 13 and its effects on fetal NPCs were observed from 3 to 72 hours after treatment (HAT), via periodic examination at six intervals. In NPCs of the fetal brain, the p53-labeling index (LI%) was markedly elevated at 3 HAT. Pyknosis and cleaved caspase-3-LI% also increased at 3 HAT, reaching peak values at 9 and 12 HAT. These parallel changes suggested the induction of apoptosis through a p53-mediated pathway. Pyknotic NPCs were distributed across the ventricular zone (VZ) of the telencephalic wall until 12 HAT, and became localized in the medial and dorsal layers at 12 and 48 HAT. Significant decreases in the numbers of mitotic NPCs and BrdU-LI% were noted from 3 HAT and 24 HAT, respectively. BrdU-positive NPCs were located in the ventral and middle layer at 24 and 48 HAT. p21-positive cells were detected at 12 and 24 HAT. The present results demonstrated that p53-mediated apoptosis was induced in all phases of the cell cycle of the NPCs in the early stage after 5-FU treatment. Furthermore, apoptosis of NPCs and suppression of cell proliferative activity are the events that take place in parallel leading to prominent reduction in the width of the telencephalic wall.     

低温处理对大蒜茎尖离体培养芽形成及生长的影响

为克服大蒜茎尖离体培养增殖率低的问题，对准备切取茎尖组织进行离体培养的大蒜种球以及切取的带有1，2片叶原基的大蒜茎尖组织(外植体)进行了低温处理，结果表明，低温处理促进了大蒜茎尖离体培养时芽的形成及生长。对外植体的低温处理比对种球的低温处理效果好，60d低温处理后，外植体低温处理区的新芽形成数是种球低温处理区的10倍，平均每个茎尖形成新芽22．2个；种球低温处理和外植体低温处理并用时。没有产生累加效应，相反，外植体低温处理的效果，被种球低温处理削弱。     

Infection dynamics of Torque teno sus virus types 1 and 2 in serum and peripheral blood mononuclear cells

This study was carried out to investigate the presence of Torque teno sus virus types 1 (TTSuV1) and 2 (TTSuV2) in a longitudinally (14 to 150 days of age) collected paired pooled sera (pSE) and peripheral blood mononuclear cells (pPBMCs) using nested polymerase chain reaction. The detection rate of TTSuV1 in pSE increased from 14 to 90 days of age, but a progressive decline was observed from 120 to 150 days of age, while in pPBMC, a high value was maintained till the end of growing-finishing period. On the contrary, except in PBMCs at 30 days of age, high detection rates of TTSuV2 were found in both pSE and pPBMCs in all sampling ages. The detection rate of TTSuVs between pSE and pPBMCs was positively correlated at all sampling ages except for TTSuV1 at 150 days of age. This is the first study showing the presence of TTSuVs in PBMCs from pigs and describing the in vivo infection dynamics of TTSuV in paired sera and PBMCs during the entire growing and finishing periods of pigs reared in conventional farms.