Epigenetic assessment of environmental chemicals detected in maternal peripheral and cord blood samples

Epigenetic alteration is an emerging paradigm underlying the long-term effects of chemicals on gene functions. Various chemicals, including organophosphate insecticides and heavy metals, have been detected in the human fetal environment. Epigenetics by DNA methylation and histone modifications, through dynamic chromatin remodeling, is a mechanism for genome stability and gene functions. To investigate whether such environmental chemicals may cause epigenetic alterations, we studied the effects of selected chemicals on morphological changes in heterochromatin and DNA methylation status in mouse ES cells (ESCs). Twenty-five chemicals, including organophosphate insecticides, heavy metals and their metabolites, were assessed for their effect on the epigenetic status of mouse ESCs by monitoring heterochromatin stained with 4￠,6-diamino-2-phenylindole (DAPI). The cells were surveyed after 48 or 96 h of exposure to the chemicals at the serum concentrations of cord blood. The candidates for epigenetic mutagens were examined for the effect on DNA methylation at genic regions. Of the 25 chemicals, five chemicals (diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se) and octachlorodipropyl ether (S-421)) caused alterations in nuclear staining, suggesting that they affected heterochromatin conditions. Hg and Se caused aberrant DNA methylation at gene loci. Furthermore, DEP at 0.1 ppb caused irreversible heterochromatin changes in ESCs, and DEP-, Hg- and S-421-exposed cells also exhibited impaired formation of the embryoid body (EB), which is an in vitro model for early embryos. We established a system for assessment of epigenetic mutagens. We identified environmental chemicals that could have effects on the human fetus epigenetic status.     

Molecular detection of Anaplasma phagocytophilum in cattle and Ixodes persulcatus ticks

The tick-borne pathogen, Anaplasma phagocytophilum (A. phagocytophilum), the causative agent of human granulocytic anaplasmosis (HGA), is increasingly becoming a public health concern as an aetiological agent for emerging infectious disease. We found A. phagocytophilum infection in a pooled sample of field-collected Ixodes persulcatus (I. persulcatus) ticks from one district in Hokkaido, Japan. Thus, to further investigate the prevalence in field-collected ticks, we used PCR assays targeting the A. phagocytophilum gene encoding 44 kDa major outer membrane protein (p44) for screening of I. persulcatus ticks and samples from cattle from pastures. Out of the 281 I. persulcatus ticks, 20 (7.1%) were found to harbor A. phagocytophilum DNA. The infection rate for A. phagocytophilum in cattle was 3.4% (42/1251). In future studies, it will be necessary to investigate effects of the infection in order to understand its pathogenesis of A. phagocytophilum in domestic animals.     

Rice phenolics efflux transporter 2 (PEZ2) plays an important role in solubilizing apoplasmic iron

Iron (Fe) is an essential micronutrient for plants; however, despite its abundance in soils it is not readily available in neutral to alkaline soils. Plants secrete phenolics, such as protocatechuic acid (PCA) and caffeic acid (CA), to absorb and utilize precipitated apoplasmic Fe from root surfaces. However, the synthesis and secretion of phenolics have not been well characterized in plants. We have identified and characterized a rice (Oryza sativa L.) mutant with reduced amounts of PCA and CA in xylem sap and root exudates; hence we named it phenolics efflux zero 2 (pez2). PEZ2 localized to the plasma membrane in onion (Allium cepa L.) epidermal cells and transported PCA when expressed in Xenopus laevis oocytes. PEZ2 expression was observed in whole root near root tips. Similarly, strong expression was observed in leaves. In line with reduced amounts of PCA and CA in xylem sap, the xylem Fe concentration was also low in pez2 plants. These results suggest that PEZ2 is involved in solubilization of apoplasmic Fe in rice.     

Effect of flutianil on the morphology and gene expression of powdery mildew

Flutianil, a fungicide effective only on powdery mildew, was previously reported to affect the host cell''s haustorial formation and nutrient absorption. Studies were conducted to investigate flutianil''s primary site of action on Blumeria graminis morphology using transmission electron microscope (TEM) observation and RNA sequencing (RAN-seq) techniques. TEM observation revealed that flutianil caused the extra-haustorial matrix and fungal cell wall to be obscured, without remarkable changes of other fungal organelles. RNA-seq analysis indicated that, unlike other powdery-mildew fungicides, flutianil did not significantly affect the constantly expressed genes for the survival of B. graminis. Genes whose expression is up- or downregulated by flutianil were found; these are the three sugar transporter genes and various effector genes, mainly expressed in haustoria. These findings indicate that the primary site of action of flutianil might be in the haustoria.     

Amphidinolide C2, New Macrolide from Marine Dinoflagellate Amphidinium Species

A new cytotoxic 25-membered macrolide, amphidinolide C2 (1), has been isolated from marine dinoflagellate Amphidinium sp. (Y-71 strain), and the structure 1 was elucidated on the basis of spectroscopic data and chemical means.     

Selective staining and visualization of hyphal sheath of a white-rot fungus <Emphasis Type="Italic">Phanerochaete crassa</Emphasis> WD1694 with phloxine B

The hyphal sheath is a morphological feature of many kinds of fungi. Although the fine structures of the sheath have been studied in detail by a number of electron microscopy techniques, the function and physiology of the hyphal sheath are not yet clarified. One reason for this is that the hyphal sheath is a colorless, mucilaginous, and delicate material so that it is not easily identified. We developed a simple method to visualize and identify the hyphal sheath of the white-rot fungus Phanerochaete crassa WD1694. The small mycelial pellets in shaken liquid cultures of P. crassa WD1694 were stained directly with phloxine B. Both the hyphae and the hyphal sheath that filled the gaps between each of the hyphae were visualized and observed by light microscopy. The stained hyphae were further studied by transmission electron microscopy, atomic force microscopy, and fl uorescence microscopy. Based on these observations, we confirmed that the staining of the hyphae was also due to the presence of the hyphal sheath that closely covered the fungal cell wall. These results clearly showed that the hyphal sheath was selectively stained with phloxine B and could be observed and identified by conventional light microscopy. Part of this report was presented at the 50th Lignin Symposium, Nagoya, October 2005     

An epidemic of parapoxvirus infection among cattle: isolation and antibody survey.

A disease characterized by papules, nodules, vesicles and, rarely, pustules and ulcers on teats was seen among cattle on a farm in Chiba Prefecture, Japan. A virus was isolated by inoculation of fetal bovine lung cell cultures from a vesicle on a teat of an infected cow. The virus was subsequently passaged in fetal bovine lung and muscle cells in which it produced complete cytopathic changes. The virus was identified by physicochemical examinations and electromicroscopic observation as a parapoxvirus. A seroepidemiological survey was performed on antibody to the isolated virus by the agar gel immunodiffusion test. The isolated virus formed a precipitation line which cross reacted with other parapoxviruses isolated previously in Japan. The positive rate was more than 50% among cattle in the Kanto district. The positive rate increased with age. It was suggested that parapoxvirus infection might have already been prevalent among cattle in Japan.     

Reproductive traits and diel activity of adultMonochamus saltuarius (Coleoptera: Cerambycidae) at two different temperatures

To investigate the reproductive traits and diel activity ofMonochamus saltuarius, 14 pairs of the adults were reared and observed at each of 20°C and 25°C. Both sexes were more active at 25°C than at 20°C irrespective of the presence or absence of light. A diel change in behavior during 24 h was found on 33% of a total of 48 observations of three age classes of both sexes. No significant difference was found in the mean longevity between sexes or between the two temperature conditions. The time required for reproductive maturation showed no significant difference within the same sex when reared at 20°C and 25°C. The mean oviposition period was significantly longer at 25°C (28.8 days) than at 20°C (12.2 days). The mean lifetime fecundity was significantly greater at 25°C than at 20°C. There was no significant difference in the oviposition ratio (number of eggs deposited/number of oviposition wounds excavated), oviposition rate or hatchability for females between the two temperature conditions.     

Estimation of kelp forest, <Emphasis Type="Italic">Laminaria</Emphasis> spp., distributions in coastal waters of the Shiretoko Peninsula,Hokkaido, Japan,using echosounder and geostatistical analysis

Sustainable management of the kelp forests of the Shiretoko Peninsula, a World Natural Heritage site, is necessary due to kelp’s ecological and economic importance. The objectives of this study were to estimate the area of kelp forests and to clarify their spatial characteristics in coastal waters of the Shiretoko Peninsula. Data on the presence/absence and thickness of kelp forests were collected via acoustic observation on transects over about 80 km using an echosounder at 200 kHz. Acoustic data were geostatistically interpolated, and the areas covered by kelp forests were estimated. Differences in kelp distribution between the eastern and western sides of the peninsula were compared. The total area of kelp forest was 3.88 km² (eastern area: 3.49 km²; western area: 0.39 km²). The range of thickness of the kelp forests was 34–91 cm. Many kelp forests in the eastern area were thick (>78 cm) and distributed continuously, while kelp forests in the western area were sparsely distributed.     

Dual effects of reversed winter–spring temperatures on year‐to‐year variation in the recruitment of chub mackerel (Scomber japonicus)

To clarify the effects of temperature on the recruitment of chub mackerel (Scomber japonicus) in the North Pacific, we investigated the influence of winter surface temperature (WST) on spawners at the time of maturity around the spawning grounds and the influence of ambient spring temperature on larvae using estimated temperature (ET) obtained from particle tracking experiments. We found a significant positive correlation between ET approximately 10 days following hatching and the recruitment per spawning stock biomass (RPS) after 2000. The closer (more meandering) the Kuroshio Current (KC) axis was in relation to the spawning ground, the higher (lower) the spring surface temperature and the higher (lower) RPS was in the spawning ground. In contrast, WST inside KC near the maturity/spawning ground was significantly negatively correlated with RPS. A significant negative correlation between the temperatures in winter and spring was detected in the area after 2000, when the conditions of the Pacific decadal oscillation index and the stability of the Kuroshio Extension were synchronous, indicating that KC shifted northward during this time. The reversed temperature pattern was consistent with the winter–spring movement of KC axis in the offshore direction and was correlated with the winter–spring difference in the intensity of the Aleutian low. These results suggest that the annual variation in chub mackerel recruitment after 2000 was strongly affected by the combined effects of ambient temperature because of the reversal of conditions that occurred between winter and spring around the maturity/spawning ground, which was related to the KC path.