Nakao Nomura

Comparative Analysis of Bacterial Diversity in Freshwater Sediment of a Shallow Eutrophic Lake by Molecular and Improved Cultivation-Based Techniques

ABSTRACT Comparative analysis of bacterial diversity in freshwater sediment collected from a shallow eutrophic lake was performed by using 16S rRNA gene clone library and improved cultivation-based techniques. Our study demonstrated that the use of gellan gum as a gelling reagent instead of agar was more effective at increasing culturability, cultivating a diverse array of novel microbes, and reducing the gaps of the results between molecular and cultivation-based analyses.

Detection of White spot syndrome virus DNA in pond soil using a 2-step nested PCR

White spot syndrome virus (WSSV) continues to be the most pathogenic among the penaeid shrimp viruses. In this study, WSSV DNA was detected in pond soil samples using a 2-step nested PCR. Primers described previously were used for the first round of amplification and based on the sequenced amplicon, an inner primer was designed for the 2nd round of amplification. Using plasmid DNA (pET 100) containing the 211 bp target WSSV sequence, analytical sensitivity showed that the 2-step nested PCR protocol was able to detect down to 0.015 fg of the plasmid DNA, or approximately 2 copies of the target DNA sequence. Persistence of WSSV DNA in pond soil samples after various time intervals was determined. WSSV-specific PCR product (161 bp) was still present in the soil samples even after 10 months of storage. The effect of soil heat treatment on the WSSV DNA was also examined. Soils were subjected to 25, 37, 50 and 70 degrees C for 1, 3 and 5 days. The results showed that PCR amplifiable WSSV DNA was still present even after 5 days at 70 degrees C. To our knowledge, this is the first report on the detection of WSSV DNA in soil samples. Based on these findings, it is concluded that the persistence of viral DNA in soil habitats may be an important aspect of WSSV ecology and may have an implication for viral transmissibility.

Growth by cell division in shrimp (Penaeus japonicus) cell culture

Abstract Tissues of Penaeus japonicus were primary cultured. Among them, dividing cells in varied shapes were found in primary cultured lymphoid cells and ovarian cells. Cell division processes of 18 lymphoid cells and 18 ovarian cells were studied. It was found that, at metaphase, aligned chromosomes of two dividing lymphoid cells and one dividing ovarian cell rotated 90° and these dividing cells cleaved along the deduced original spindle axis, which suggest that these cells may have carried on asymmetric division-like cell division. These results prove that the culture conditions used are suitable for cultured lymphoid cells and ovarian cells of P. japonicus to proliferate in vitro and that cultured lymphoid cells and ovarian cells still have the ability to carry on cell division. These findings will give a light to the study of continuous shrimp cell line establishment and will also provide us with the knowledge of shrimp cell division.

PENAEID (PENAEUS JAPONICUS) LYMPHOID CELLS REPLICATE BY CELL DIVISION IN VITRO

SummaryPenaeid cell culture has gained much attention as a potential model to facilitate researches on the characterization of the virus and to develop more sophisticated and improved diagnostic procedures for use in the aquaculture industry. However, to date, cell division processes of cultured penaeid cells have not been found, which is suggested as one of the reasons that block the establishment of the continuous penaeid cell lines. We reported here the cell division processes of cultured lymphoid cells of Penaeus japonicus. The culture medium used was based on M199 and was modified by supplementing saline components. Cultures were incubated at 25°C, and 5% CO2 was supplemented. In primary cultured lymphoid cells, dividing cells in different shapes were found. Cell division processes of 12 dividing lymphoid cells were tracked. After cell division, their daughter cells turned into fibroblast-like or epithelioid cells. These results proved that the culture conditions used were suitable for lymphoid cells of P. japonicus to proliferate in vitro and that cultured lymphoid cells still had the ability to carry out cell division. These findings would give light to the establishment of continuous penaeid cell lines and would also provide us with the knowledge of cell division processes of the penaeid.

Analysis of the relationship between luminescence and toxicity of Vibrio carchariae pathogenic to shrimp

Aquaculture deterioration is caused by Vibrio harveyi, which is one of the main shrimp pathoges. Vibrio carchariae having the same phenotypic characteristics as V. harveyi was isolated from a shrimp farm and used in this study to investigate the relationship between luminescence and toxicity. Luminescence was verified by monitoring the expression of the LuxR gene. It was found that the relationship between the expression of LuxR and toxicity was the most significant factor related to luminescence. Results showed that LuxR expression was highest after 20 h of culture, which is comparable to the highest scintillation counter luminescence value (20 h). However, toxin levels in the culture broth were highest between 24 h-36 h of culture. Significant decrease in the toxicity level was observed after 36 h. It was also found that there was a decrease in toxicity level after 12 h of incubation at room temperature of the V. carchariae culture supernatant. The effect of the addition of cell-free culture supernatant on the luminescence of V. carchariae was also determined. Results showed that the addition of cell-free supernatant from 24 h-old culture was most effective in inducing and maintaining luminescence.

Study on the relationship of protease production and luminescence in Vibrio harveyi

Aims:  To demonstrate that Vibrio harveyi produces various types of toxins and how the production of those toxins is related with luminescence.

Improved MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for the measurement of viable animal cell number in porous cellulose carriers

A method to measure cell concentration in porous cellulose carriers was developed. Results of the comparison of 5 colorimetric assays showed that MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay by the solubilization of the formazan with DMSO(dimethyl sulfoxide) was the most suitable method for the measurement of cell concentration in porous cellulose carriers. The growth of CHO-K1 (Chinese hamster ovary)-immobilized in different types of carrier was evaluated.

The effect of copper concentration on the virulence of pathogenic Vibrio harveyi

Aim:  To demonstrate the influence of copper on luminescence and toxin production in Vibrio harveyi.

Expansion of human autologous cytotoxic T lymphocytes on fixed target tumor cells

Human tumor specific cytotoxic T lymphocytes (CTL) were expanded on formalin-fixed autologous target tumor cells derived from glioblastoma multiforme. Growth response of the CTL restimulated with the fixed target cells was larger than those with live target cells. The results suggest that formalin-fixed tumor cells will be stable sources of tumor antigen for efficient autologous CTL expansion and be useful for adoptive immunotherapy of tumors.

Production of human growth hormone by protein-free cultivation of anchorage-dependent cells with porous cellulose carrier

Abstract Verots S3 cells derived from Vero-317 cells which can grow in biotin-containing MEM medium were successfully cultured in a protein-free medium with a porous cellulose carrier. The growth of Verots S3 cells without a carrier was inhibited because they easily aggregated under excessive shear stress, but the cells grew very quickly when cultured with the porous cellulose carrier. This improvement in cell growth was thought to be due to the protection from fluid shear stress afforded by the porous structure of the carrier. Production of human growth hormone (hGH) by repeated batch cultivation of Verots S3 cells was much better with than without the carrier. Cultivation with a temperature shift from 39 to 33°C gave hGH production up to 400% higher in a spinner culture with the cellulose carrier. For large-scale cultivation, temperature sensitive Verots S3 cells were cultivated with the porous cellulose carrier in a rotary column bioreactor and the perfusion culture results showed that the cell density inside the carrier increased up to 7.9 × 10 8 cells·ml-carrier −1 in 2 weeks. No concentration gradient of the cells in the rotary column was observed.