Proteome analysis: a new approach to identify key proteins involved in acquisition of maturational competence and oocyte maturation of medaka oocytes

We have applied analytical methods of proteomics to identify key proteins that are involved in physiologically important processes on fish reproduction. We have improved methods of proteome analysis for fish oocytes and revealed changes of expression patterns of oocyte proteins and protein phosphorylation during acquisition of maturational competence and signal transduction of oocyte maturation of medaka oocytes.     

Growth hormone secretion during longterm incubation of the pituitary of the Japanese eel,Anguilla japonica

Growth hormone (GH) secretion from organ-cultured pituitaries of the eel (Anguilla japonica) was studied during incubation in a defined medium for 2 weeks, using a homologous radioimmunoassay which does not distinguish between the two molecular forms of eel GH. The total amount of GH secreted increased gradually during the incubation period; so that the amount of GH released on day 14 was about 30 times greater than that on day 1. On day 14, the proportion of GH released relative to the total amount of GH present (the sum of GH released into the medium and residual content in the pituitary) was 96% and the amount produced on day 14 was 4 times greater than the content in the unincubated pituitary. Somatostatin (SRIF, 1.8 × 10^-7 M) inhibited the increase in GH release. On day 7, the proportion of GH released by pituitaries treated with SRIF (28%) was less than that released by the control pituitary (91%). There was no significant difference in GH release between the pituitaries incubated in isotonic medium (300 mOsm) and those in hypotonic medium (240 mOsm) for 2 weeks except for the first 3 days, when the pituitaries in hypotonic medium secreted significantly greater amounts of GH than those incubated under isotonic condition. Hypertonic medium (350 mOsm) had no effect on GH release except for significant inhibition on days 6 and 14. When secretion of the two forms of GH (GH I and II) was examined after separation by polyacrylamide gel electrophoresis followed by densitometry, slightly more GH I tended to be secreted than GH II during the culture period, although the effects of SRIF and osmolality of the media on GH I release were similar to those on GH II. It is concluded that GH secretion and production in the eel is mainly under the inhibitory control of hypothalamus, and that osmolality has a minimum influence on the GH release.     

Cyanobacteria from Terrestrial and Marine Sources Contain Apoptogens Able to Overcome Chemoresistance in Acute Myeloid Leukemia Cells

In this study, we investigated forty cyanobacterial isolates from biofilms, gastropods, brackish water and symbiotic lichen habitats. Their aqueous and organic extracts were used to screen for apoptosis-inducing activity against acute myeloid leukemia cells. A total of 28 extracts showed cytotoxicity against rat acute myeloid leukemia (IPC-81) cells. The design of the screen made it possible to eliminate known toxins, such as microcystins and nodularin, or known metabolites with anti-leukemic activity, such as adenosine and its analogs. A cytotoxicity test on human embryonic kidney (HEK293T) fibroblasts indicated that 21 of the 28 extracts containing anti-acute myeloid leukemia (AML) activity showed selectivity in favor of leukemia cells. Extracts L26-O and L30-O were able to partly overcome the chemotherapy resistance induced by the oncogenic protein Bcl-2, whereas extract L1-O overcame protection from the deletion of the tumor suppressor protein p53. In conclusion, cyanobacteria are a prolific resource for anti-leukemia compounds that have potential for pharmaceutical applications. Based on the variety of cellular responses, we also conclude that the different anti-leukemic compounds in the cyanobacterial extracts target different elements of the death machinery of mammalian cells.     

Detection of urinary luteinizing hormone in Japanese black cows after administration of gonadotropin-releasing hormone

The blood luteinizing hormone (LH) surge in cows is well studied. However, little is known about urinary LH in cows. This study examined urinary LH concentrations after administration of gonadotropin-releasing hormone (GnRH) in six Japanese black cows to induce LH secretion from the pituitary gland into the bloodstream. Abrupt rises in plasma and urinary LH were observed after GnRH administration. Plasma and urinary LH peaked at 2 and 5 hr, respectively. A positive correlation was observed between plasma LH concentrations and urinary LH amounts. Ovulation was confirmed in the cows after 48 hr of GnRH administration. These data strongly suggest that urinary LH is derived from plasma LH, which triggers ovulation in cows.     

Determination of polybrominated diphenyl ethers and polybrominated dibenzo-p-dioxins/dibenzofurans in marine products

Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants in plastics and textile coatings, and these compounds have been recognized as ubiquitous environmental contaminants. Furthermore, it is considered a serious problem that polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/DFs), having toxicities similar to those of chlorinated dioxins, are generated by the manufacture of brominated flame retardants (BFRs) such as PBDEs, and formed by the combustion of substances containing BFRs. Several congeners of PBDD/DFs and PBDEs have been detected in the adipose tissue of the Japanese. Although food is suspected as an exposure source, little information is available regarding the levels of these brominated compounds in food, as compared with information regarding dioxin or polychlorinated biphenyls. It is necessary to investigate the levels of these brominated organic compounds in various foods and to estimate their influence in the case of human exposure. We developed an efficient method of analyzing PBDEs and PBDD/DFs contents in food samples using accelerated solvent extraction and determined the concentrations in several marine products such as raw fish, processed foods, and seaweed purchased in Japan. A recovery test (n = 5) using the method and involving dried fish showed acceptable recoveries of 57.7-78.5% (RSD 5.4-15.9%) for PBDEs and 50.0-56.4% (RSD 1.5-7.9%) for PBDD/DFs. In the analysis of marine product samples, several congeners of PBDEs were detected in raw fish, processed fish, and seaweed; the highest concentration of sigmaPBDEs was detected in yellowtail (1161 pg/g whole basis), followed by mackerel (553.5 pg/g whole basis). The most dominant congener present in these marine samples was 2,2',4,4'-tetraBDE (#47).     

Antioxidant activities of black and yellow soybeans against low density lipoprotein oxidation

Several studies have demonstrated that the daily intakes of soy foods were associated with a reduced cardiovascular risk. The aim of our study was to investigate the inhibitory effect of black soybeans on low density lipoprotein (LDL) oxidation in comparison to yellow soybeans. The extract from black soybean had a longer LDL oxidation lag time than that from yellow soybean (205 +/- 16 and 65 +/- 3 min, respectively). When both soybeans were divided into the seed coat and the mixture of the germ and cotyledon, the diluted extract solution from the black soybean seed coat prolonged the lag time significantly more than the original extract of the yellow soybean seed coat. On the other hand, antioxidant effects of the extract from the mixture of germs and cotyledons were similar in both soybeans. Regarding total polyphenol contents, the seed coat of black soybean had a higher polyphenol content than that of yellow soybean (29.0 +/- 0.56 and 0.45 +/- 0.02 mg/g, respectively). Interestingly, the mixture of the germ and cotyledon hydrolyzed by beta-glucosidase in both soybeans showed a stronger inhibitory effect on LDL oxidation than that before being hydrolyzed by beta-glucosidase. These results suggest that black soybeans may be more effective in inhibiting LDL oxidation than yellow soybeans because of total polyphenols contents in its seed coat. In addition, aglycones, which are rich in soybeans fermented or hydrolyzed by beta-glucosidase, may play a crucial role in the prevention of oxidation-related diseases.     

Forest soil community responses to plant growth-promoting rhizobacteria and spruce seedlings

The influence of plant-growth-promoting rhizobacteria (PGPR) and spruce seedlings on the composition and activity of forest soil microbial communities was studied in a microcosm experiment in which sterile, sand-filled 25mm×150mm glass tubes were treated with a forest soil suspension containing Bacillus or Pseudomonas PGPR and 2-week-old spruce seedlings. Eighteen weeks after treatments were established, bacterial, actinomycete and fungal population sizes were determined by dilution plating, as were seedling dry weights and soil carbon substrate utilization profiles using Biolog plates. PGPR inoculation had little influence on the population sizes of actinomycetes or fungi. However, significant effects were detected on the total bacterial population size, primarily in microcosms without seedlings. Euclidean distances between treatments plotted on two dimensions by multidimensional scaling showed that the introduction of PGPR strains changed the type of microbial community, particularly when inoculated into soil without seedlings. Significant changes were also detected in one soil type in the presence of seedlings. Our results suggest that the type of soil community and the presence of seedlings are significant factors influencing the responses of soil communities to bacterial inoculation, and that for some soil communities, the presence of seedlings may mitigate perturbations caused by the introduction of PGPR. Received: 24 February 1997     

A model to estimate available timber and forest biomass and reforestation expenses in a mountainous region in Japan

We developed a model to estimate supply potentials and available amounts of timber and forest biomass resources from profitable sub-compartments of thinning and final felling operations. Economic balances were estimated while considering not only harvesting expenses but also reforestation expenses after final felling, which should be considered for sustainable forest management. Harvesting expenses were estimated based on two types of timber harvesting systems and three types of forest biomass harvesting systems in each sub-compartment. Then, the model was applied to Nasushiobara city of Tochigi prefecture, Japan. Reforestation expenses had large negative impacts on the financial balances of final felling operations. Few sub-compartments were profitable after considering reforestation expenses. Most profitable sub-compartments were those with mechanized operation systems and landing sales. These accounted for 17.19% of all sub-compartments, while only 5.75% of the sub-compartments were profitable based on their current operation systems and landing sales. Although the overall supply potentials of timber and forest biomass resources were 380,000 m3 and 210,000 Mg, respectively, and 15 times the planned harvest of coniferous tree volume of 25,000 m3year-1 and 50 times the annual demand for the woody gasification power generation of 4,000 Mg year-1 in Nasushiobara, available amounts of timber and forest biomass resources were only49,429 m3 and 33,333 Mg, which were 13.0% and 15.7% of supply potentials for landing sales with mechanized operation systems.