Tomoaki Itayama

Detection and sequencing of the microcystin LR-degrading gene, mlrA, from new bacteria isolated from Japanese lakes

mlrA is the only microcystin-degrading gene detected in Sphingomonas sp. MJ-PV. The gene has an extremely rare nucleotide sequence and homologous genes have not yet been discovered in the DNA database. We discovered the existence of a gene homologous to mlrA in new microcystin-degrading bacteria, MD-1 and Y2. These strains possessed mlrA homologues, and the identities of the genes of MD-1 and Y2 with the corresponding MJ-PV exceeded 98% and 84%, respectively. On the other hand, the mlrA gene was not detected in laboratory strains of the closely related Sphingomonas spp. strains employing hemi-nested polymerase chain reaction detection using two primer sets. Although the microcystin-degrading bacteria were closely related strains, they did not cluster together as the same species. We can conclude that the mlrA gene is conserved in three different bacterial species, and it is unique to microcystin degraders but not to the genus Sphingomonas.

Detection of hormone active chemicals using genetically engineered yeast cells and microfluidic devices with interdigitated array electrodes

Endocrine disruptors that act like hormones in the endocrine system might have toxic effects. Therefore, it is important to develop a portable device that can detect hormone active chemicals in samples rapidly and easily. In this study, a microfluidic device was developed for the detection of hormone active chemicals using genetically engineered yeast cells. The yeast cells were used as biosensors since they were genetically engineered to respond to the presence of hormone active chemicals by synthesizing β‐galactosidase (β‐gal). For achieving further sensitivity, we incorporated interdigitated array (IDA) electrodes (width, 1.2 μm; gap, 0.8 μm) with 40 electrode fingers into the analytical chamber of the microfluidic device. The yeast cells precultured with a hormone active chemical, 17β‐estradiol (E2), were trapped from the main channel of the device to the analytical camber by electrophoresis. After trapping in the analytical chamber, we performed electrochemical detection of β‐gal induced in the yeast cells with the IDA electrodes. Actually, electrochemical detection was performed on p‐aminophenol that was converted from p‐aminophenyl‐β‐D‐galactopyranoside with β‐gal. The electrochemical signals from the yeast cells precultured with 17β‐estradiol were successfully detected with the device. Furthermore, the inhibitory effects of antagonists such as tamoxifen were also detected electrochemically by using the device. Thus, the present microfluidic device can be used for highly sensitive detection of hormone active chemicals.

Electrophoretic Cell Manipulation and Electrochemical Gene-Function Analysis Based on a Yeast Two-Hybrid System in a Microfluidic Device

A novel microfluidic device with an array of analytical chambers was developed in order to perform single-cell-based gene-function analysis. A series of analytical processes was carried out using the device, including electrophoretic manipulation of single cells and electrochemical measurement of gene function. A poly(dimethylsiloxane) microstructure with a microfluidic channel (150 microm in width, 10 microm in height) and an analytical chamber (100 x 20 x 10 microm (3)) were fabricated and aligned on a glass substrate with an array of Au microelectrodes. Two microelectrodes positioned in the analytical chamber were employed as a working electrode for the electrophoretic manipulation of cells and electrochemical measurements. A yeast strain ( Saccharomyces cerevisiae Y190) carrying the beta-galactosidase reporter gene was used to demonstrate that the device could detect the enzyme. Target cells flowing through the main channel were introduced into the chamber by electrophoresis using the ground electrode laid on the main channel. When the cell was treated with 17beta-estradiol, gene expression was triggered to produce beta-galactosidase, catalyzing the hydrolysis of p-aminophenyl-beta- D-galactopyranoside to form p-aminophenol (PAP). The enzymatically generated PAP was detected by cyclic voltammetry and amperometry at the single-cell level in the chamber of the device. Generator-collector mode amperometry was also applied to amplify the current response originating from gene expression in the trapped single cells. After electrochemical measurement, the trapped cells were easily released from the chamber using electrophoretic force.

Detection of cyprinid herpesvirus-3 DNA in lake plankton.

The disease caused by cyprinid herpesvirus-3 (CyHV-3) severely impacts the natural freshwater ecosystem and damages carp and koi farming, however, the pathway of CyHV-3 transmission remains unclear. It is possible that the virus adheres to plankton, which then facilitate viral movement and transmission, and therefore, it is hypothesised that plankton are involved in the disease dynamics. In this study, plankton were collected at eight sites in the Iba-naiko lagoon; we detected and quantified CyHV-3 DNA from plankton samples. The results of the correlation analysis showed a significant positive correlation between CyHV-3 copies and the number of Rotifera, suggesting that CyHV-3 binds to and/or is concentrated by Rotifera. Our results suggest that plankton affect viral ecology in the natural environment.

Biodegradation of microcystis and microcystins by indigenous nanoflagellates on biofilm in a practical treatment facility

Abstract The potential for degradation of Microcystis and microcystins by organisms in biofilm in a practical biological treatment facility combined with conventional treatment processes was examined. Viable cells of Microcystis viridis, which produced microcystin LR, RR and YR, were degraded in 6 to 10 days by the addition of biofilm collected in early summer, midsummer and autumn. Monas sp. grew remarkably well compared with other organisms in the samples of all seasons. Therefore, we conclude that Monas sp. was indigenous animals in the biological treatment facility and was able to degrade Microcystis cells. Some previous studies reported the predation of Microcystis cells by Monas guttula in pure culture conditions (monoxenic culture). We clarified that Monas sp. can grow and prey on Microcystis cells even in mixed culture including many kinds of other organisms in biofilm in a practical treatment facility. Also, intracellular microcystin LR, RR and YR in Microcystis cells could be degraded simultaneously with high reduction of Microcystis cells by organisms in biofilm.

Stress response to daily temperature fluctuations in common carp, Cyprinus carpio L.

The littoral zone of lakes and lagoons is often used by fish for feeding or reproduction. However, the large changes in temperature that are typical of natural environments, including the littoral zone, represent a potential stressor for fish. Despite the importance of this habitat, little is known about the effect of daily temperature fluctuations on the stress responses of fish. We monitored daily temperature changes in the near-shore and offshore regions of a natural lagoon between May and July 2008–2010. We observed large temperature fluctuations more frequently in the near-shore zone than the offshore zone. We then exposed common carp (Cyprinus carpio) to a temperature regime similar to that observed in the near-shore zone and measured the levels of cortisol released into the water. The rate of cortisol release increased when carp were exposed to an increase in temperature of ~0.6°C/h over a 5-h period. Conversely, there was no change in the rate of release when temperatures decreased. Our results highlight the importance of maintaining high temporal resolution when evaluating the stress response to daily fluctuations temperature.

USE OF FLUE GAS DESULFURIZATION GYPSUM FOR THE REMOVAL OF OFF-FLAVOR COMPOUNDS IN FISH POND WATER

Accumulation of off-flavor in fish flesh caused by the presence of geosmin and 2- methylisoborneol (MIB) in pond waters is a worldwide quality problem in aquaculture. This study investigated the effectiveness of synthetic flue gas desulfurization gypsum, (FGDG) a cheap and readily soluble material from coal-fired electric utilities, for the removal of off flavor substances: geosmin and MIB, in fish pond water. Water samples from fish ponds were spiked with known concentrations of geosmin and MIB and varying amounts of FGDG (0, 200, 400 and 600 mg/L) were added to compare the removal rates of geosmin and MIB, chlorophyll a levels and orthophosphate. The experiment was conducted for 9 days. It was revealed that FGDG had significantly reduced both geosmin and MIB in spiked pond waters when compared with the control (0 mg/L) (P 0.05) different from 600 mg/L. The highest removal rate of MIB (75.63%) was found with the treatment with 400 mg/L CFG after 6 days. In addition, 200 mg/L FGDG was also highly effective in reducing chlorophyll a and orthophosphate concentration through calcium phosphate precipitation. It was suggested that 200 mg/L FGDG is the suitable dose to apply for the removal of off-flavor compounds in fish pond waters.

Environment-KHV-carp-human linkage as a model for environmental diseases

To predict outbreaks of infectious disease and to prevent epidemics, it is essential not only to conduct pathological studies but also to understand the interactions between the environment, pathogen, host and humans that cause and spread infectious diseases. Outbreaks of mass mortality in carp caused by Cyprinid herpesvirus 3 (CyHV-3), formerly known as koi herpesvirus (KHV), disease have occurred worldwide since the late 1990s. We proposed an environment–KHV–carp–human linkage as a conceptual model for “environmental diseases” and specify research subjects that might be necessary to construct and shape this linkage.

Nonlinear Systems far from Equilibrium and Missing Route to “Living State”

Process of regeneration of a hydra from dissociated cell aggregate was studied. The process induces cell-sorting period, cavity-formation period and structure forming period. Simultaneously, nerve network formation takes place, apparently induced by head structure formation. The aggregate changes from initially disordered cell assembly to an ordered animal within a few days. Measured quantities related to the transition are presented. Difference between the living state and known dynamical states of nonlinear system far from equilibrium is discussed.

Self-organization of structures and appearance of functions in biological systems

As a part of effort to physically define what is the living state, we describe process of regeneration of a hydra animal from a tendem graft and a dissociated cell aggregate. Particularly, a transition from a dissociated cell aggregate which is nothing but a mechanically gathered cells to an animal which is an organically interacting cell system is, in a sense, similar to an nonequilibrium system undergoing a transition to an ordered state. Also a fundamental difference between measurement of the physical systems and the biological systems are discussed.