Acoustic evaluation of the vertical distribution of dwarf ayu Plecoglossus altivelis altivelis in Lake Biwa

SUMMARY: The vertical distribution of dwarf ayu Plecoglossus altivelis altivelis in the pelagic waters of Lake Biwa was evaluated from June to September in 1995–97 from eight acoustic surveys using a quantitative echosounder. In each survey, echoes from a depth range of 3 m to the sea bottom were collected at a station every 2.7 s for 24 h together with measurements of vertical profiles of water temperature and chlorophyll-a. The ayu's echoes were identified using an underwater video camera. The ayu were observed near the maximum chlorophyll-a depth and above the thermocline. Their density was highest at depths of 4–11 m with 2–4 individuals/m ³ and was almost zero below 20 m. Echo signs were recorded as having a frequent duration of more than 1 h at night, whereas were of a shorter duration in the day. The fish stay in the epilimnion during the day without any clear vertical migration, but are distributed more uniformly at night. The advantages of remaining in the epilimnion are discussed in terms of food availability and predator avoidance.     

Occurrence and Distribution of Perfluorooctane Sulfonate and Perfluorooctanoic Acid in the Rivers of Tokyo

Comprehensive survey of major rivers in the Tokyo metropolitan area was conducted for clarifying the emission sources of perfluorooctane sulfonate (PFOS) in Tokyo. PFOS was found at all sampling sites at concentrations ranging from 0.5 to 58 ng L^?1; in addition to this, it was also indicated that unknown PFOS emission sources are present in the midstream of the Tama River basin. The relationship between PFOS and perfluorooctanoic acid (PFOA) was constant at a ratio of 10:3 (PFOS/PFOA) throughout the Tama River basin. The sum of daily load amounts of PFOS from Tokyo’s major rivers to Tokyo Bay reached 215 g day^?1. This value corresponds to 12.8 μg day^?1 per person using the sum of wastewater treatment district populations. In contrast, an estimation of PFOS contribution of domestic wastewater was also attempted, and the contribution was 1.6 μg day^?1 per person. We took samples up trunk sewers in the Tama River and finally found at the highest PFOS concentration (58,000 ng L^?1) from one of the wastewater of the electronic parts manufacturing facilities.     

Na?ve-like conversion enhances the difference in innate in vitro differentiation capacity between rabbit ES cells and iPS cells

Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use.     

Specific detection of potentially allergenic kiwifruit in foods using polymerase chain reaction

Kiwifruit (Actinidia deliciosa and Actinidia chinensis) is allergenic to sensitive patients, and, under Japanese regulations, it is one of the food items that are recommended to be declared on food labeling as much as possible. To develop PCR-based methods for the detection of trace amounts of kiwifruit in foods, two primer pairs targeting the ITS-1 region of the Actinidia spp. were designed using PCR simulation software. On the basis of the known distribution of a major kiwifruit allergen (actinidin) within the Actinidia spp., as well as of reports on clinical and immunological cross-reactivities, one of the primer pairs was designed to detect all Actinidia spp. and the other to detect commercially grown Actinidia spp. (i.e., kiwifruit, Actinidia arguta, and their interspecific hybrids) except for Actinidia polygama. The specificity of the developed methods using the designed primer pairs was verified by performing PCR experiments on 8 Actinidia spp. and 26 other plants including fruits. The methods were considered to be specific enough to yield target-size products only from the target Actinidia spp. and to detect no target-size products from nontarget species. The methods were sensitive enough to detect 5-50 fg of Actinidia spp. DNA spiked in 50 ng of salmon testis DNA used as a carrier (1-10 ppm of kiwifruit DNA) and 1700 ppm (w/w) of fresh kiwifruit puree spiked in a commercial plain yogurt (corresponding to ca. 10 ppm of kiwifruit protein). These methods would be expected to be useful in the detection of hidden kiwifruit and its related species in processed foods.     

Microdroplet In Vitro Fertilization Can Reduce the Number of Spermatozoa Necessary for Fertilizing Oocytes

Successful in vitro fertilization (IVF) in mice has been achieved using spermatozoa at concentrations specifically optimized for the experimental conditions, such as species and source of spermatozoa. Although IVF in mice is mostly performed using about 80–500 µl drops, it is expected that the number of spermatozoa used for insemination can be reduced by decreasing the size of the IVF drops. The present study was undertaken to examine the extent to which the number of spermatozoa used for IVF could be reduced by using small droplets (1 µl). We devised the experimental parameters using frozen–thawed spermatozoa from C57BL/6 mice in anticipation of broader applications to other mouse facilities. We found that as few as 5 spermatozoa per droplet could fertilize oocytes (1 or 3 oocytes per droplet), although the fertilization rates were low (13–15%). Practical fertilization rates (> 40%) could be achieved with frozen-thawed C57BL/6J spermatozoa, which are sensitive to cryopreservation, when 20 sperm per droplet were used to inseminate 3 oocytes. Even with spermatozoa from a very poor quality suspension (10% motility), about 25% of oocytes were fertilized. Our calculations indicate that the number of inseminated spermatozoa per oocyte can be reduced to 1/96–1/240 by this method. In two separate embryo transfer experiments, 60% and 47%, respectively, of embryos developed to term. Our microdroplet IVF method may be particularly advantageous when only a limited number of motile spermatozoa are available because of inadequate freezing-thawing or genetic reasons.     

Antitumor activity of compounds isolated from leaves of Eriobotrya japonica

In a search for possible antitumor agents from natural sources, megastigmane glycosides and polyphenolic constituents isolated from the leaves of Eriobotrya japonica (Rosaceae) were found to inhibit the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of Epstein-Barr virus early antigen in Raji cells. Roseoside and procyanidin B-2 were among the active compounds found in an in vitro assay; these compounds were further assessed for antitumor activity in vivo in a two-stage carcinogenesis assay on mouse skin. Roseoside significantly delayed carcinogenesis induced by peroxynitrite (initiator) and TPA (promoter), and its potency was comparable to that of a green tea polyphenol, (-)-epigallocatechin 3-O-gallate, in the same assay.     

Aureusidin synthase: a polyphenol oxidase homolog responsible for flower coloration

Aurones are plant flavonoids that provide yellow color to the flowers of some popular ornamental plants, such as snapdragon and cosmos. In this study, we have identified an enzyme responsible for the synthesis of aurone from chalcones in the yellow snapdragon flower. The enzyme (aureusidin synthase) is a 39-kilodalton, copper-containing glycoprotein catalyzing the hydroxylation and/or oxidative cyclization of the precursor chalcones, 2',4',6',4-tetrahydroxychalcone and 2',4',6',3,4-pentahydroxychalcone. The complementary DNA encoding aureusidin synthase is expressed in the petals of aurone-containing varieties. DNA sequence analysis revealed that aureusidin synthase belongs to the plant polyphenol oxidase family, providing an unequivocal example of the function of the polyphenol oxidase homolog in plants, i.e., flower coloration.     

Differential detection of shrimp and crab for food labeling using polymerase chain reaction

Shrimp and crab are well-known as allergenic ingredients. According to Japanese food allergy labeling regulations, shrimp species (including prawns, crayfishes, and lobsters) and crab species must be differentially declared when ≥10 ppm (total protein) of an allergenic ingredient is present. However, the commercial ELISA tests for the detection of crustacean proteins cannot differentiate between shrimp and crab. Therefore, two methods were developed to discriminate shrimp and crab: a shrimp-PCR method with postamplification digestion and a crab-PCR method that specifically amplifies a fragment of the 16S rRNA gene. The sensitivity and specificity of both PCR methods were verified by experiments using DNA extracted from 15 shrimp species, 13 crab species, krill, mysid, mantis shrimp, other food samples (cephalopod, shellfish, and fish), incurred foods, and commercial food products. Both PCR methods could detect 5 pg of DNA extracted from target species and 50 ng of genomic DNA extracted from incurred foods containing 10 ppm (μg/g) total protein of shrimp or crab. The two PCR methods were considered to be specific enough to separately detect species belonging to shrimp and crab. Although false-positive and false-negative results were obtained from some nontarget crustacean species, the proposed PCR methods, when used in conjunction with ELISA tests, would be a useful tool for confirmation of the validity of food allergy labeling and management of processed food safety for allergic patients.