Electrode System for the Determination of Microbial Populations

Determinations of microbial populations were carried out by using a new electrode system composed of two electrodes. Each electrode was constructed from a platinum anode and a silver peroxide cathode. The anode of the reference electrode was covered with cellulose dialysis membrane. The response time of the electrode system was 15 min in culture broth, and current differences between the two electrodes were proportional to populations of microbial cells in cultures of Saccharomyces cerevisiae and Lactobacillus fermentum. Current differences were reproducible; the average relative error was 5%. Furthermore, cell populations of S. cerevisiae in a fermentor could be continuously estimated by using this electrochemical method.     

GGC and StuI polymorphism on the androgen receptor gene in endometrial cancer patients

Androgens have an anti-proliferative effect on endometrial cells. Human androgen receptor (AR) gene contains two polymorphic short tandem repeats of GGC and CAG, and a single-nucleotide polymorphism on exon 1 that is recognized by the restriction enzyme, StuI. Prior studies have shown that the lengths of the CAG repeat are inversely and linearly related to AR activity and associated with endometrial cancer. However, little is known about the GGC repeat and the StuI polymorphism of the AR gene. Thus, we investigated whether these AR polymorphisms are risk factors for endometrial cancer. To test this hypothesis, the genetic distributions of these polymorphisms were investigated in blood samples from endometrial cancer patients and healthy controls. The allelic and genotyping profiles were analyzed by polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and direct DNA sequencing, and analyzed statistically. The GGC repeat was significantly longer in endometrial cancer patients as compared to normal healthy controls. In general, an increased risk of endometrial cancer was found with increasing GGC repeat. The relative risk for the 17 GGC repeat was greater than 4, as compared to controls. However, the StuI polymorphism was not significantly different between patients and controls. The findings suggest that increased numbers of GGC repeat on the AR gene may be a risk factor for endometrial cancer.     

Improvement of a mediator-type biochemical oxygen demand sensor for on-site measurement.

We characterized a mediator-type biochemical oxygen demand (BOD) sensor with a three-electrode system using potassium ferricyanide (FC) and Pseudomonas fluorescens in our previous study. In the present study, we have utilized the advantages of a mediator-type biosensor, which does not require air-supply equipment for on-site measurements, and made a fully disposable sensor tip for a portable device. The tip consists of a two-electrode system with P. fluorescens immobilized on a cellulose acetate membrane and is packaged in polyester film to prevent it from drying out. By aeration with a 0.1 M NaCl solution of P. fluorescens (after growth), the sensor responses as well as their reproducibility and stability have been successfully improved. The responses increased more than seven times, and the calibration curve from 15 to 260 mg l(-1) also remained linear although the response decreased approximately half the original after at least 35 days in storage. The reproducibility of the sensor responses improved to 12.7% (average of relative standard deviations (RSDs)) in the calibration curve obtained by using the Organization for Economic Cooperation and Development synthetic sewage. Examination of real samples from three different sources showed that the BOD as determined by the sensor correlates well with the conventional 5-day BOD method (r(2)=0.982, 0.823, and 0.809). Consequently, the aeration process makes it possible to realize rapid, and in situ measurements without the long conditioning process that is generally required to activate the microorganisms immobilized on bio-films before use. Finally, we have designed a portable device that utilizes our disposable sensor tip.     

Quantitative analysis of polymerase chain reaction using anisotropy ratio and relative hydrodynamic volume of fluorescence polarization method.

The method based on the combination of polymerase chain reaction (PCR) and fluorescence polarization is presented. A targeted DNA was amplified with a 5'-fluorescein labeled primer, using a 256 bp DNA fragment of stx2 gene in Escherichia coli O157:H7 (188-443 bp) as a template. The fluorescence anisotropy of the 5'-fluorescein labeled primer increased upon the polymerization through Taq polymerase. The conversion of primer to PCR product was quantitatively monitored by anisotropy ratio and relative hydrodynamic volume. This system was also applied to the determination of E.coli O157:H7.     

In vivo fluorometric method for early detection of cyanobacterial waterblooms

A specific method was developed for monitoring the concentration of cyanobacteria (blue-green algae) before waterblooms, based on their characteristics ofin vivo fluorescence. The excitation and emission spectra of cyanobacteria are very different from those of eukaryotic algae, due to the importance of phycocyanin, rather than chlorophylla, in determining the fluorescence characteristics. Our results, based on four cyanobacteria:Microcystis aeruginosa, Anabaena cylindrica, Phormidium tenue andSpirulina platensis, indicate that excitation at 620 nm and its emission at 645 nm is a sensitive and specific method for their detection. Furthermore, the addition of 10 M photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) induced only 3% increase in phycocyanin fluorescence, suggesting that this measurement is almost independent of the ongoing rate of photosynthesis.Author for correspondence     

Cloning and expression of Citrobacter freundii hydrogenase genes in Escherichia coli

Summary Escherichia coli mutants deficient in hydrogenase activity (Hyd^-) were derived from E. coli C600 by mutagenesis with nitrosoguanidine. Hydrogenase activities of mutant strains; HK-2, HK-7, HK-8, HK-16, HK-23, and HK-26 were below 1/100 that of the parental strain E. coli C600. Conjugational transfer of plasmid F-143 to the mutants was carried out and hydrogenase activities of the transformants were assayed. Recovery of hydrogenase activities in mutant strains; HK-2, HK-7, HK-8, HK-16, and HK-23 was observed, but not for HK-26. Two kinds of hydrogenase genes of Citrobacter freundii were cloned on pBR 322 and hybrid plasmids pCBH2 and pCFH1 were obtained. Hydrogenase activities of mutant strains HK-2, HK-8 and HK-16 were complemented with pCBH2 and strain HK-7 with pCFH1 respectively. The other mutant strains, HK-23, HK-26, however, were not complemented with these plasmids.     

DNA cleavage based on high voltage electric pulse

A high voltage electric pulse was applied to DNA cleavage. The DNA cleavage reaction was dependent on the voltage amplitude, pulse number and pulse width. Radical scavengers and ESR data indicated the possibility that active species such as OH radical were strongly related to DNA cleavage.     

Seasonal and site-specific variability in terrigenous particulate organic carbon concentration in near-shore waters of Lake Biwa, Japan

Identifying sources of particulate organic matter (POM) is important for clarifying fundamental mechanisms by which lake food webs are sustained. We determined carbon and nitrogen stable isotope ratios of POM in near-shore waters of Lake Biwa, a large, meso-eutrophic lake in Japan, to estimate relative contributions of terrigenous particulate organic carbon (T-POC), plankton-derived POC (P-POC) and epilithon-derived POC (E-POC) to POC in near-shore waters. Samples were collected during different months (November, February, May and July) at 29 sites located near the mouth of tributary rivers with different discharge and catchment land use. The data revealed that POC mainly consisted of P-POC and T-POC, with relative contributions varying widely over season and among locations. E-POC generally contributed little to the near-shore POC. Path analyses revealed that the concentration of riverine POC whose isotopic signatures were similar to those of rice straws increased with a larger %paddy field area in the catchment of tributary rivers, which subsequently enhanced T-POC inputs to near-shore waters through riverine transportation. Furthermore, our results suggested that T-POC contribution was influenced, with a time lag, by wave-driven turbulence and shore topography, which appear to affect sedimentation and resuspension of T-POC.     

Physical structure of habitat network differently affects migration patterns of native and invasive fishes in Lake Biwa and its tributary lagoons: stable isotope approach

A good understanding of how migratory animals use their habitat network is expected to provide important insights for the prediction of population dynamics at both local and regional scales. We focused on how the physical structure of a habitat network could affect fish migration between Lake Biwa and its tributary lagoons. Although the lagoons provide suitable breeding and nursery grounds for native fishes, it is a matter of concern that they can also be a hotbed of invasive exotic fishes. Here, we assessed the migration patterns of native crucian carps (Carassius spp.) and exotic largemouth bass (Micropterus salmoides) and bluegill sunfish (Lepomis macrochirus) using their carbon and nitrogen stable isotopes as migration tracers. As there were marked differences in the carbon isotope ratios of basal food webs between the main lake and its tributary lagoons, stable isotopic signatures of individual fishes collected from each lagoon enabled us to judge whether they were residents of the lagoon or recent immigrants from the main lake. The analysis revealed that native and invasive fishes showed different migration patterns across a variety of lagoons. Exotic fishes frequently immigrated from the main lake to the lagoon as the distance of the channel connecting these two habitats was short. For native crucian carps, in contrast, their migrations were unaffected by the channel distance but were promoted by narrow channels. Physical barriers of weirs and dense vegetation within the channel obstructed their migrations. Such ecological information on migration behavior will be vital to plan designs for habitat restoration to conserve native fishes.     

Restoration of Escherichia coli hyd B Mutant Hydrogenase by Cultivation in Media Containing Various Metals

Hydrogenase restoration of Escherichia coli hydrogenase deficient mutant HK7, which carries a mutation at hyd B locus, was studied. Anaerobic growth of HK7 in the presence of iron chloride or vanadium chloride resulted in the restoration of hydrogen uptake activity of hydrogenase, but not hydrogen evolution activity. The growth of HK7 in the presence of nickel chloride restored total hydrogenase activity (hydrogen uptake and evolution) as Waugh and Boxer (1986) reported. Therefore, the leniency of HK7 hyd B product might permit the transportation and incorporation of iron chloride or vanadium chloride in hydrogenase, resulting in the alteration of hydrogenase activity.