Tokyo Bay, a eutrophic bay in Japan, receives nutrients from wastewater plants and other urban diffuse sources via river input. A transect was conducted along a line from the Arakawa River into Tokyo Bay to investigate the ecological relationship between the river outflow and the distribution, abundance and population structure of ammonia-oxidizing bacteria (AOB). Five surficial marine sediments were collected and analysed with polyphasic approaches. Heterogeneity and genetic diversity of beta-AOB populations were examined using restriction fragment length polymorphism (RFLP) analysis of 16S rRNA and amoA genes. A shift of the microbial community was detected in samples along the transect. Both 16S rRNA and amoA genes generated polymorphisms in the restriction profiles that were distinguishable at each sampling site. Two 16S rRNA gene libraries were constructed using the reverse transcription polymerase chain reaction (RT-PCR) method to determine the major ammonia oxidizers maintaining high cellular rRNA content. Two major groups were observed in the Nitrosomonas lineage; no Nitrosospira were detected. The effort to isolate novel AOB was successful; the isolate dominated in the gene libraries. For quantitative analysis, a real-time PCR assay targeting the 16S rRNA gene was developed. The population sizes of beta-AOB ranged from 1.6 x 10(7) to 3.0 x 10(8) cells g(-1) in dry sediments, which corresponded to 0.1-1.1% of the total bacterial population. An immunofluorescence staining using anti-hydroxylamine oxidoreductase (HAO) antibody was also tested to obtain complementary data. The population sizes of ammonia oxidizers ranged between 2.4 x 10(8) and 1.2 x 10(9) cells g(-1) of dry sediments, which corresponded to 1.2-4.3% of the total bacterial fraction. Ammonia-oxidizing bacteria cell numbers deduced by the two methods were correlated (R = 0.79, P < 0.01). In both methods, the number of AOB increased with the distance from the river mouth; ammonia-oxidizing bacteria were most numerous at B30, where the ammonium concentration in the porewater was markedly lower and the nitrite concentration was slightly higher than nearby sites. These results reveal spatial distribution and shifts in the population structure of AOB corresponding to nutrients and organic inputs from the river run-off and phytoplankton bloom.

A polyphasic, culture-independent study was conducted to investigate the abundance and population structure of ammonia-oxidizing bacteria (AOB) in canal sediments receiving wastewater discharge. The abundance of AOB ranged from 0.2 to 1.9% and 1.6 to 5.7% of the total bacterial fraction by real-time PCR and immunofluorescence staining, respectively. Clone analysis and restriction endonuclease analysis revealed that the AOB communities influenced by the wastewater discharge were dominated by Nitrosomonas, were similar to each other, and were less diverse than the communities outside of the immediate discharge zone.Tokyo Bay is a representative enclosed eutrophic bay in the southern Kanto region of Japan. More than 86% of the shoreline is reclaimed, and artificial structures, such as canals and landfill islands, occupy the inner bay area (29). These artificial structures often hamper water exchange and accelerate precipitation of sludge containing excess organic matter, heavy metals, and toxic compounds (16,17,29). These human activities negatively affect the sea-bottom environment and limit natural remediation in the coastal ecosystem (19).Nitrification is essential to the nitrogen cycle in aquatic environments. When coupled with denitrification and/or anaerobic ammonium oxidation, it relieves the negative impacts of eutrophication through removal of nitrogen to the atmosphere as nitrous oxide or molecular nitrogen (5, 9, 38). However, nitrification is sensitive to environmental stress and contaminants (18, 23, 37). Ammonia-oxidizing bacteria (AOB) carry out the first, rate-limiting step of nitrification: conversion of ammonia to nitrite. The ecology and physiology of AOB are particularly difficult to study by conventional cultivation techniques because of their long generation times and low growth rates, which can result in underestimations of their numbers in the environment (21). Compared to the studies of AOB diversity in nature (1,4,6,12,13,22), quantitative studies have been limited, especially for marine environments (11). Therefore, a rapid, culture-independent detection technique for AOB would be useful for the study of marine AOB. Through this study, we developed and combined molecular and novel immunofluorescence staining approaches to investigate the spatial distribution, abundance, and population structure of AOB in a canal area of Tokyo Bay that is heavily polluted by excess organic and nutrient loading from wastewater treatment plant discharges. Particularly important for this study was quantification of AOB because of the deficiency of quantitative studies of AOB populations in marine environments

Physicochemical properties of two types of adriamycin preparation, suspensions and emulsions prepared for i.a. chemotherapy of hepatocellular carcinoma, were investigated. A suspension was prepared by dispersing adriamycin directly into the lipid contrast medium, Lipiodol, whereas an emulsion was obtained by emulsifying an aqueous solution of adriamycin into Lipiodol. The dispersibility of the drug in each preparation was examined microscopically. The chemical stability of and drug release from the preparation were determined by high-performance liquid chromatography and spectrophotometry, respectively. The suspension was then given to ten patients with primary hepatocellular carcinoma. The suspension maintained good dispersibility without coagulation of drug particles, whereas coalescence of aqueous droplets and the resultant phase separation occurred 4 h after preparation of the emulsion. Both preparations maintained the initial drug content for at least 1 week at room temperature. The release of adriamycin was more prolonged in the suspension than in the emulsion. After i.a. administration of the suspension, a selective accumulation of Lipiodol in the tumor and decrease in serum alpha-fetoprotein (AFP) levels were found in most patients. A significant amount of adriamycin was still detected in hepatic specimens resected from two patients 1 and 2 months after treatment. These findings suggest that the adriamycin-Lipiodol suspension may be a useful preparation for targeting chemotherapy to hepatocellular carcinoma.

An organic extract of airborne particles collected in Tokyo and its fractions (neutral, acidic, and basic) were investigated in Ames Salmonella assays for mutagenicity and in newborn mice for carcinogenicity. Mutagenicity to TA100 and TA98 strains was detected in the whole extract, the neutral, the acidic and the basic fractions with and without metabolic activation. In the carcinogenicity test, the incidence of lung tumor was as follows: whole extract, 4/25; neutral fraction, 7/25; acidic fraction, 0/20; basic fraction, 1/11; vehicle, 2/39; and uninjected, 3/47. The neutral fraction of the extract of the airborne particles showed highly potent mutagenicity and a high incidence of lung tumors in mice.

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