Life cycle of <Emphasis Type="Italic">Dilophus okamurae</Emphasis> (Phaeophyceae) and its associated invertebrate fauna in Onagawa Bay,Japan

ABSTRACT:   The life cycle of Dilophus okamurae and the associated benthic invertebrate fauna were studied monthly or bimonthly from June 1998 to September 1999 at a depth of 4–7 m in Onagawa Bay along the Pacific coast of north-eastern Honshu, Japan (38°28'N, 149°29'E). Dry biomass ranged from 230 g/m² in June to 10 g/m² in October. Sporophytes with tetrasporangia appeared during July to September 1998. Gametophytes with oogonia or antheridia were observed for the first time in May 1999 during observation. The absence of mature sporophytes in 1999 suggests that the life cycle is completed over two years. The sympatric gastropods Lirularia iridescens , Homalopoma sangrarense, Conotalopia minima and Cantharidus callichroa synchronized with seasonal changes in biomass of this algae. In contrast, Barleeia angustata and B. trifasciata fluctuated independently. The juvenile sea urchin Strongylocentrotus nudus appeared in the algal population starting in October at 0.7 mm mean test diameter at high density, and disappeared after March.     

Analyses of leaves from open field-grown transgenic poplars overexpressing xyloglucanase

The transgenic expression of Aspergillus xyloglucanase cDNA (AaXEG2) with 35S promoter in the leaves of open field-grown poplars was studied. The level of xyloglucan in the transgenic poplars was decreased to 15–16% in the non-fertile soil (forest-field soil) and to 21–22% in the fertile soil (farming-field soil) compared with that of the wild-type poplars. The leaves exhibited a smaller surface area with more rounded teeth than those of the wild-type plants, similar to the sun leaf variety that was grown in the incubation room and subsequently greenhoused. The majority of total veins with water-conducting vascular bundles were shorter in the leaves of the transgenic poplars than those of the wild type. This decrease in vein length may result from a decrease in xyloglucan during leaf development, from which large numbers of proteins were markedly downregulated in the leaves of the transgenic plants via proteomic analysis. It seems likely that the leaves of the transgenic poplars came to relax the edges of their tooth rather than extend their veins as a result of the loosening of the xyloglucan cellulose networks in the leaves.     

Effects of (−)‐Epigallocatechin Gallate on the Motility and Penetrability of Frozen–Thawed Boar Spermatozoa Incubated in the Fertilization Medium

Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis) and is known for its antioxidant effects. The objective of the present study was to examine the effects of EGCG during in vitro fertilization (IVF) on the sperm quality and penetrability into oocytes. In the first experiment, the effects of concentration and incubation period of EGCG on the motility and penetrability of spermatozoa were examined. When frozen–thawed spermatozoa were incubated in IVF medium supplemented with 0 (control), 1, 50 and 100 μm EGCG for 1, 3 and 5 h, supplementation with 50 and 100 μm EGCG improved motility of the spermatozoa (p < 0.05), but not viability, as compared with the control group. When frozen–thawed spermatozoa were co‐incubated with in vitro‐matured (IVM) oocytes in IVF medium supplemented with 50 and 100 μm EGCG for 5 h, supplementation of EGCG had positive effects on sperm penetration rates. In the second experiment, the effects of supplementation of EGCG in IVF medium on penetrability of sperm from different boars and development of fertilized oocytes were evaluated. When frozen–thawed spermatozoa from six boars were co‐incubated with IVM oocytes in IVF medium supplemented with 50 μm EGCG, the effect of EGCG on sperm penetration and development of oocytes after fertilization was found to vary with individual boar. Our results indicate that motility and penetrability of boar spermatozoa are improved by co‐incubation with 50 μm EGCG, but the effects vary with individual boars.     

Retrospective study of porcine circovirus 2 infection in Japan: seven cases in 1989

Retrospectively, we demonstrated that seven pigs necropsied in 1989 had characteristic porcine circovirus 2 (PCV-2) lymphoid tissue lesions of severe lymphoid depletion, syncytial giant cell formation, and intracytoplasmic inclusion bodies in macrophages. Immunohistochemically, PCV-2 antigen was detected in these tissues and the distribution of positive staining corresponded to the distribution of inclusion bodies. Electron microscopy demonstrated viral particles compatible with porcine PCV-2. Therefore, the disease occurred in Japan as early as 1989.     

Identification of Cylindrocladium sp. causing damping-off disease of Japanese black pine (Pinus thunbergii) and factors affecting the disease severity in a black locust (Robinia pseudoacacia)-dominated area

The regeneration of Japanese black pine (Pinus thunbergii) seedlings is inhibited in a black locust (Robinia pseudoacacia)-dominated area. We examined the presence of pathogenic fungi in Japanese black pine seedlings in the area in order to determine the effect of pathogenic fungi on the inhibition of regeneration. When Japanese black pine seedlings were planted in the soil obtained from a black locust-dominated area, all of the seedlings died under low-intensity light conditions, whereas 84% of the seedlings survived in the soil obtained from a Japanese black pine-dominated area under the same light conditions. One fungus was isolated from 48.7% of the dead pine seedlings and was identified as Cylindrocladium pacificum Kang, Crous & Schoch, based on the morphological characteristics, growth, and DNA analysis. This fungus was also isolated from 50% of the dead pine seedlings in 2005 and 66.7% of the seedlings in 2006—both were planted in a black locust-dominated area. The virulence of this fungus increased under high-nitrogen and/or low-intensity light conditions. These results reveal the possibility that the soil eutrophication and shading by the black locust are conducive to a severe damping-off disease and threaten the survival and regeneration of Japanese black pine seedlings.     

Morphological changes in the rat endocrine pancreas within 12 h of intravenous streptozotocin administration

We examined early morphological changes in pancreatic endocrine cells within 12 h of intravenous streptozotocin (STZ) administration (60 mg/kg). Thirty rats were allocated either to a control group (vehicle alone) or to one of four experimental groups tested after 3, 6, 9 and 12 h. Karyopyknosis and cytoplasmic vacuoles were first observed in beta-cell cytoplasm 3 h after STZ administration (STZ-3 h), and the most severe damage was found in beta cells at STZ-12 h. Insulin-positive non-islet cells were observed near the intercalated duct (ICD) and/or centroacinar (CA) cells at STZ-6 h and their numbers peaked at STZ-6 h. The distribution patterns of the insulin-positive cells and those of nestin and insulin-like growth factor-1 were similar and their nuclei were positive for proliferating cell nuclear antigen. Thus, ICD cells and/or CA cells reacted immediately to transform into insulin-secreting cells to replace injured beta cells (or to compensate for the lack of beta cells) within 12 h of STZ administration.     

Neutralizing test of hemagglutinating encephalomyelitis virus (HEV) in FS-L3 cells cultured without serum

FS-L3 cells, originating from porcine kidney, were used for propagation of Hemagglutinating encephalomyelitis virus (HEV) and development of a virus neutralizing (VN) test. Sera of pigs, rats, cows and dogs had VN activities to HEV. On the other hand, sera of mice, rabbits, goats, sheep, horses, cats, chickens, hamsters and human did not have measurable VN activities, although these sera had high HI activities. Our results support the idea that the VN is a more reliable measure of HEV infection than the conventionally used HI test.     

Effect of the Removal of Cumulus Cells on the Nuclear Maturation, Fertilization and Development of Porcine Oocytes

The present study was conducted to investigate the effects of attachment of cumulus cells to porcine oocytes during the process of maturation and fertilization on the nuclear maturation, fertilization and subsequent development after in vitro fertilization (IVF). In the first experiment, the cumulus cells were removed from cumulus-oocyte complexes (COCs) at 0, 24 and 42 h after the onset of maturation culture and were then cultured until reaching 42 h of cultivation. In the second experiment, COCs were denuded as described in the first experiment, then fertilized and cultured for 7 days. As a control, cumulus cells were allowed to maintain attachment to the oocytes until the end of IVF. The proportion of oocytes reaching metaphase II significantly increased with the delay in the removal treatment of cumulus cells. The proportion of normal fertilization gradually increased with delay in the removal treatment of cumulus cells from COCs until the end of IVF. However, no significant difference in the proportion of normal fertilization was found between the 42-h and control groups. The removal treatment of cumulus cells in the 0- and 24-h group significantly (p < 0.05) decreased the proportion of cleaved embryos when compared with the control, and none of them developed to the blastocyst stage. The proportion of development to the blastocyst stage was significantly higher (p < 0.05) in the control group than in the 42-h group (18.1% vs 12.4%; p < 0.05). The present study indicates that the attachment of cumulus cells to the oocyte during maturation and fertilization is important to support oocyte nuclear maturation, fertilization and subsequent embryo development. Particularly, the attachment of cumulus cells to the oocyte during IVF promotes embryonic development.     

Analysis of DNA Fragmentation of Porcine Embryos Exposed to Cryoprotectants

The chemical toxicity of cryoprotectants to porcine embryos was examined by the evaluation of survival and DNA damage after exposure to cryoprotectants. Porcine blastocysts were exposed to 10% of ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GLY) for 1 h at room temperature (23-25 degrees C) and then cultured in vitro for 24 h. The survival rates of blastocysts exposed to PD and GLY were significantly lower than those of control blastocysts in which the embryos were exposed to carrier solution without cryoprotectants. Significantly more DNA-fragmented nuclei occurred in the cryoprotectant-exposed blastocysts, compared with the control blastocysts. Moreover, the indices of DNA-fragmented nuclei in the blastocysts without blastocoele re-formation after culture were significantly higher than those with blastocoele re-formation, irrespective of the exposure treatment. These results indicate that the exposure of porcine blastocysts to cryoprotectant decreases the survival rates and increases the DNA-fragmented nuclei in embryos.