Characterization and expression of a P-450-like mycinamicin biosynthesis gene using a novel Micromonospora-Escherichia coli shuttle cosmid vector

A 29 kb shuttle cosmid vector, pTYS507, was constructed from a cryptic Micromonospora griseorubida plasmid and the Escherichia coli cosmid pJB8. Subcloning of mycinamicin II biosysnthesis genes in pTYS507 led to the identification of a DNA region that could complement a mutant of M. griseorubida that lacked both hydroxylase and epoxidase activities. Nucleotide sequence and mutational analysis suggested that a single P-450-like protein catalyzes both reactions.     

Bird carcass detection from integrated trials at multiple wind farms

It is often necessary to estimate the number of wind turbine collision fatalities to assess impacts to birds following construction of wind farms. Detection of bird carcasses at wind turbines in the field is affected by carcass persistence and searcher detection rate. Integrated detection trials, which integrate carcass persistence and searcher detection trials into the periodic fatality search, have been proposed as an effective method for estimating these parameters. The purpose of our study was to test whether and how environmental factors affect integrated detection trial outcomes at multiple wind farms. We conducted this study at 10 wind farms in various environments of Japan. Binary data on trial outcomes in open versus forested areas served as our response variable in a generalized additive mixed model informed by days into trial, carcass body mass, season, whether snow covered the ground, and precipitation. For both ground cover types, days into trial and body mass were included in all the top models, suggesting that these factors most influenced bird carcass detection probability in integrated trials. The best model in open areas included days into trial, body mass, snow, and precipitation, and the best model in forested areas included days into trial, body mass, snow, precipitation, and season. Values of area under the curve indicated high accuracy of the best model for both ground cover types. The survey design needs to be appropriate to the size of the target species and to the environment in which the impacts will occur, such as the site's seasonality, its ground cover, and whether snow will cover the ground. Frequency of post-construction fatality monitoring should also be set cautiously, especially at wind farms located on small-bird migration routes, at wind farms in open areas, in areas with snow-covered ground in winter, or in forested areas during spring and summer because detection probabilities decline fastest under such conditions.     

Efficient production of anti-(hepatitis B virus) antibodies and their neutralizing activity in chimpanzees

 For industrial production of human monoclonal antibodies (hmAb) against hepatitis B virus surface antigen (HBsAg), we scaled-up a short-term perfusion culture in serum-free medium, which was chosen as the most suitable culture method, to a 50-l fermentor equipped with a rotating shear filter. Using hydrophobic chromatography as the initial step of hmAb purification, the mAb HBW4, HBW6 and W471 were isolated in good quality from the respective culture broths in yields of approximately 75%. Each of the three purified hmAb alone, and a cocktail of the three, protected chimpanzees against HB virus, when injected intravenously 3 h after viral challenge, as long as the serum antibody levels were significant. A pharmacokinetic study using cynomolgus monkeys demonstrated that the hmAb have a long plasma half-life and bioavailability of approximately 76% upon intramuscular injection in primates. Thus, anti-HBsAg hmAb produced by an industrial process are expected to be successfully used in clinical fields. Received: 20 June 1994/Received revision: 16 September 1994/Accepted: 10 October 1994     

Chromosomes of Temnocephala minor,an ectosymbiotic turbellarian on Australian crayfish found in Kagoshima Prefecture,with karyological notes on exotic turbellarians found in Japan

Records of exotic turbellarian species found in Japan are reviewed from taxonomic and karyological viewpoints. Temnocephala minor Haswell, 1888, an ectocommensal on a freshwater crayfish of Australia, was found from culture ponds of Cherax tenuimanus (introduced from W. Australia) in Kagoshima Prefecture. T. minor had the chromosome number of 2x = 18 (2sm + 2m + 2m + 2sm + 2m + 2m + 2m + 2sm + 2m). The following 3 species of exotic freshwater triclads were recorded from tanks and ponds used for tropical fish culture: Dugesia austroasiatica Kawakatsu, 1985 (2x = 16), Dugesia tigrina (Girard, 1850) (2x = 16) and Rhodax? sp. (3x = 24; 3x = 24 &; 3x + 1LB + 1SB = 25 + 1SB). The following 3 species of exotic terrestrial triclads were recorded: Bipalium nobile Kawakatsu et Makino, 1982 (2x = 10), Bipalium kewense Moseley, 1878 (2x = 18), and Platydemus manokwari de Beauchamp, 1962 (n = 6, 2x = 12). An extensive occurrence of P. manokwari in the Southwest Islands of Japan may be due to an unexpected introduction of the animal in very recent years.     

Changes in eicosapentaenoic acid content of Navicula saprophila, Rhodomonas salina and Nitzschia sp. under mixotrophic conditions

Three species of microalgae able to produce eicosapentaenoic acid(EPA) were collected from brackish and sea water around Japan. The species were identified as Navicula saprophila, Rhodomonassalina and Nitzschia sp. EPA as a proportion of total fatty acids increased in the presence of acetic acid for Rhodomonas salina and Nitzschia sp. However, Navicula saprophila displayed the greatest productivity of EPA and the EPA content of its biomass was enhanced under mixotrophic conditions in the presence of acetic acid. This revised version was published online in August 2006 with corrections to the Cover Date.     

Kinetics and collagenolytic role of eosinophils in chronic gastric ulcer in the rat

 An association between eosinophils and tissue damage has been observed in numerous disorders. However, few reports have addressed the role of infiltrating eosinophils in gastric ulcer healing. The aim of this study was to investigate the kinetics and role of eosinophils infiltrating experimental chronic gastric ulcers in the rat. We developed a monoclonal antibody against human matrix metalloproteinase 1 (MMP1) purified from conditioned culture medium of human skin fibroblasts. Acetic acid-induced gastric ulcers were resected from rats on days 1, 3, 5, 10, 20, 40, and 180 after the days of induction (day 0). Tissue specimens were immunostained with this antibody and examined with an electron microscope. Few eosinophils were observed in the granulation tissue until day 20. By days 40 and 180, MMP1-positive eosinophils had increased in the granulation tissue of open ulcers. Azan staining revealed dispersed collagen fibers around infiltrating eosinophils. In contrast, scars demonstrated few eosinophils in fibrous tissue on days 40 and 180. Eosinophils which express MMP1 infiltrate granulation tissue at the chronic stage of gastric ulceration. The results suggest that eosinophils may play a role in tissue remodeling and deterioration of ulceration. Accepted: 18 March 1997     

Over-production of Thermus protease in dense culture of Escherichia coli using two carbon sources

Escherichia coli JM109(DE3) harboring expression plasmid pkAQNÆC30, which carries the Thermus protease aqualysin I (AQI) gene, was cultivated with glucose as a sole carbon source. The final cell concentration was over 15 g dry weight/l and the amount of AQI produced reached approximately 130 kU/ml broth. Moreover, by using two carbon sources, glucose and glycerol, the production yield was increased to over 200 kU AQI/ml, while suppressing the formation of inhibitory acetic acid.     

Sequence analysis of a gene cluster involved in metabolism of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia AC1100.

Burkholderia cepacia AC1100 utilizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. PT88 is a chromosomal deletion mutant of B. cepacia AC1100 and is unable to grow on 2,4,5-T. The nucleotide sequence of a 5.5-kb chromosomal fragment from B. cepacia AC1100 which complemented PT88 for growth on 2,4,5-T was determined. The sequence revealed the presence of six open reading frames, designated ORF1 to ORF6. Five polypeptides were produced when this DNA region was under control of the T7 promoter in Escherichia coli; however, no polypeptide was produced from the fourth open reading frame, ORF4. Homology searches of protein sequence databases were performed to determine if the proteins involved in 2,4,5-T metabolism were similar to other biodegradative enzymes. In addition, complementation studies were used to determine which genes were essential for the metabolism of 2,4,5-T. The first gene of the cluster, ORF1, encoded a 37-kDa polypeptide which was essential for complementation of PT88 and showed significant homology to putative trans-chlorodienelactone isomerases. The next gene, ORF2, was necessary for complementation and encoded a 47-kDa protein which showed homology to glutathione reductases. ORF3 was not essential for complementation; however, both the 23-kDa protein encoded by ORF3 and the predicted amino acid sequence of ORF4 showed homology to glutathione S-transferases. ORF5, which encoded an 11-kDa polypeptide, was essential for growth on 2,4,5-T, but the amino acid sequence did not show homology to those of any known proteins. The last gene of the cluster, ORF6, was necessary for complementation of PT88, and the 32-kDa protein encoded by this gene showed homology to catechol and chlorocatechol-1,2-dioxygenases.     

Origin of mitotic cells of the chorionic villi in direct chromosome analysis

Summary The origin of mitotic cells was investigated histologically in chorionic tissue, metaphase plates of which were used for direct cytogenetic study. Mitotic figures were often observed in the Langhans' cell layers, but absolutely none were seen in the syncytium and stromal cells.