Ken-ichi Suga

Magnetic and transport characteristics on high Curie temperature ferromagnet of Mn-doped GaN

Mn-doped GaN films on sapphire (0001) substrates were grown by molecular beam epitaxy system using ammonia as nitrogen source. The result of magnetization measurement gives Curie temperature as high as 940 K. The field and temperature dependencies of the magnetization show coexistence of ferromagnetic and paramagnetic phases. In addition, the temperature dependencies of electrical resistance and carrier concentration were measured to investigate the relation between the ferromagnetism and transport property. Below about 10 K, a similar anomalous increase of magnetization and resistance is observed.

Effect of methanol concentration on the production of human β2-glycoprotein I domain V by a recombinant Pichia pastoris: A simple system for the control of methanol concentration using a semiconductor gas sensor

Abstract The methylotrophic yeast Pichia pastoris is one of the best hosts for the production of foreign proteins because of the presence of the strong AOX1 promoter induced by methanol. Methanol feeding during the production phase of the foreign proteins is important because methanol not only induces protein production but also provides energy source for the host cells. Excess methanol inhibits the growth of host cells, while an insufficient amount of energy source and/or methanol starvation lead to poor growth and production. We constructed a simple methanol control system consisting of a semiconductor gas sensor and a relay. Using this system, we studied the effect of methanol concentration on the production of a model foreign protein, human β2-glycoprotein I domain V. The methanol concentrations were kept constant at 1.5, 10, 17, or 31 g·l−1 (±5%) during the production phase. Although the specific rates of growth and methanol consumption decreased with increase in the methanol concentration, the specific production rates increased, indicating that the energy for the production competed with that for cell growth. Accordingly, we provided glycerol as an extra energy source during the production phase, with the result that the specific production rate increased two times. Our simple and inexpensive system will help bioengineering studies on the production of recombinant proteins in P. pastoris, the growth and production of objective proteins in which are dependent on the methanol concentration.

Amplified Gene Location in Chromosomal DNA Affected Recombinant Protein Production and Stability of Amplified Genes

Previously, we established an easy and quick construction method for obtaining a stable and highly productive gene‐amplified recombinant Chinese hamster ovary (CHO) cell line. With a gradual increase in methotrexate (MTX) concentration, gene‐amplified cell pools had high and stable specific growth and production rates. Moreover, the phenotype of gene‐amplified cells seemed to be affected by the location of the amplified gene in chromosomal DNA. We suspected that various kinds of gene‐amplified cells might appear during the long‐term selection to construct gene‐amplified cell pools. To clarify the behavior of gene‐amplified cell pools during a stepwise increase of MTX concentration, we isolated gene‐amplified clones derived from gene‐amplified cell pools. We compared the characteristics of isolated clones, such as the productivity of recombinant protein, stability of amplified genes, and the location of amplified genes. As a result, telomere‐type clones, in which the amplified gene was located near the telomeric region, were found to be more stable and productive than other types of clones. Telomere‐type clones had over 100 copies of amplified genes in the chromosomal DNA. In contrast, a large number of other types of clones had less than 10 copies of amplified genes. During long‐term cultivation in the absence of MTX, in other types of clones, amplified genes rapidly decreased in the chromosomal DNA.

Effect of amino acids on glutathione production by Saccharomyces cerevisiae

SummaryThe constituent amino acids of the glutathione (GSH) tripeptide chain, glutamate, cysteine and glycine, were investigated for positive effects on GSH production in shake-flask cultures of Saccharomyces cerevisiae with glucose as the carbon source. Cysteine was confirmed as the key amino acid for increasing the specific GSH production rate, ϱg, but showed some growth inhibition, especially in the second growth phase (ethanol-assimilation phase). An intracellular cysteine delivery agent, thiazolidine, showed a similar pattern of increased GSH production and growth inhibition, but to a slightly lesser degree, compared with free cysteine. The initial cysteine concentration affected both the specific growth rate, µ, and ϱg, up to about 5 mm for µ and about 2–3 mm for ϱg. Results of the [35S]cysteine-labelling experiments suggest a complicated role of cysteine in increasing GSH production and further investigation may be necessary.

Cysteine addition strategy for maximum glutathione production in fed-batch culture of Saccharomyces cerevisiae

SummaryA good cysteine addition method that could increase the specific glutathione (GSH) production rate (G9G) was investigated and utilized to maximize total GSH production in fed-batch culture of Saccharomyces cerevisiae. The single-shot addition of cysteine was a better method compared to a continuous method that maintained a constant cysteine concentration in the reactor. The shot method increased ϱG about twofold compared to a culture without cysteine. The increase in ϱG by the shot method can be achieved without growth inhibition if the cysteine dose is maintained at 0.7 mml·g-1 cell or less. The positive effect on ϱG (at every specific growth rate, µ) was saturated when the cysteine shot concentrations was 3 mM or more. A simple model was developed consisting of mass balance equations and the relationship between µ and ϱG, for the single cysteine shot addition method. From this model an optimal operating strategy was determined to maximize total GSH production in fed-batch culture. This optimal operation consisted of separating the process into phases of (1) cell growth and (2) GSH production, through a bang-bang profile control of µ, and a shot of cysteine just at the start of the GSH production phase. In other words, the cysteine shot time and the µ switching time should be the same. For a total feeding time of 10 h, both the switching time of µ and cysteine shot time were calculated to be about 6.4 h.

Evaluation of stable and highly productive gene amplified CHO cell line based on the location of amplified genes

In order to establish an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, variouskinds of stepwise methotrexate (MTX) selection were carriedout. The specific growth and production rates of the cell were compared with each other, and the distribution of the amplified gene location was determined using fluorescence in situ hybridization (FISH). The specific growth andproduction rates of the cell pool reached the highest levels under the selection condition in which the stepwise increase in the MTX concentration was most gradual; about 82% of amplified genes were observed near the telomeric region. During long-term cultivation without MTX, the percentage ofamplified genes near the telomeric region hardly changed, butthat of amplified genes at other regions decreased. Based on these results, stable and highly productive cell pools could be easily and quickly constructed and amplified and gradual stepwise increase of the MTX concentration. In addition, the FISH technique was powerful tool to evaluate highly productiveand stable gene-amplified cells based on the chromosomal location of the amplified gene.

The enhancement of specific antibody production rate in glucose- and glutamine-controlled fed-batch culture.

The concentration effects of certain amino acids (Asp, Ile, Leu, Lys, Met, Val, Phe and Gln which were highly consumed during cultivation), and glucose on cell growth and antibody productivity were investigated using dish culture. From these experiments, it was found that only glutamine enrichment enhanced the specific antibody production rate. The other amino acids described above did not affect either the specific growth rate or specific antibody production rate. Thus we investigated the quantitative effects of glutamine concentration in the range of 0.4∼33.3 mmol·1−1 on kinetic parameters in fed-batch culture which kept both glucose and glutamine concentration constant. As a result the specific growth rate decreased with increase in glutamine concentration in the range larger than 20 mmol·1−1. The specific antibody production rate had a maximum value at about 25 mmol·1−1 glutamine concentration.

Kinetic study of poly-D(-)-3-hydroxybutyric acid (PHB) production and its molecular weight distribution control in a fed batch culture of Alcaligenes eutrophus

Abstract The kinetics of poly- d (−)-3-hydroxybutyric acid (PHB) production was studied in Alcaligenes eutrophus H16 (ATCC 17699) fed-batch culture using butyric acid as a sole carbon source. In order to control the butyric acid concentration at a steady value, a constant butyric acid feeding policy was developed. At a butyric acid concentration of 3 g/ l , pH 8.0, the maximum values of specific PHB production rate, production yield, and PHB content in the cell were obtained. The effects of the cultivation conditions on the molecular weight distribution of PHB were also studied. The average molecular weight distribution decreased as the butyric acid concentration increased. When a nitrogen source was added to the fermentation medium after adequate PHB accumulation in the cells, not only PHB production but also degradation occurred simultaneously. The degradation rate of low molecular weight polymer was greater than that of high molecular weight polymer, and as a result the average molecular weight shifted to the higher value.

In vivo estimation of bioartificial liver with recombinant HepG2 cells using pigs with ischemic liver failure.

Biological efficacy of a recombinant human hepatic cell line, glutamine synthetase transfected HepG2 (GS-HepG2), was examined with large-scale culture in a circulatory flow bioreactor and in pigs with ischemic liver failure. GS-HepG2 cells were cultured in a circulatory flow bioreactor from 5 × 107 to 4 × 109 cells for 109 days. The cells showed ammonia removal activity even under substrate (glutamic acid)-free medium, suggesting that the GS catalyzed the activity using intracellular glutamic acid that had been pooled during conventional culture. When GS-HepG2 bioartificial liver (BAL) was applied to pigs with ischemic liver failure, survival time was prolonged to 18.8 ± 6.1 h (mean ± SD, n = 4) from 13.8 ± 5.4 h (n = 6) and 10.7 ± 4.1 h (n = 6) (groups treated with cell-free BAL and treated with plasma exchange and continuous hemodia-filtration, respectively). Laboratory data indicated the tendency for improvement in increase of blood ammonia level and decline of blood coagulation indices in the GS-HepG2 BAL-treated group. The advantages and potential for the cell line as a bioreactor in BAL is also discussed, comparing to those of isolated porcine hepatocytes.

Long-term culture of glutamine synthetase-transfected HepG2 cells in circulatory flow bioreactor for development of a bioartificial liver.

Glutamine synthetase (GS) is involved in an accessory pathway of ammonia removal in mammals. To develop a bioartificial liver with a human cell line, GS gene was transfected into HepG2 cells, which had no ammonia removal activity. After culturing in the presence of methionine sulfoximine (MSX), a GS inhibitor, we obtained a MSX-resistant HepG2 subline (GS-HepG2), which had amplified GS gene; ammonia removal activity was estimated to be 1/7 of that of rat primary culture hepatocytes. The cells were cultured in a circulatory flow bioreactor for 109 days, while they multiplied from 5 × 107 to 4 × 109 cells. Three days after inoculation, the ammonia level of the culture medium was lowered to a level maintained thereafter, suggesting that using recombinant cell lines for bioartificial livers enables long-term repeated treatment for hepatic failure patient. Judging from the rate of decrease in the amount of the added ammonia, the ammonia removal capability of 4 × 109 GS-HepG2 cells was almost equivalent to 5 × 108 porcine hepatocytes inoculated into the circulatory flow bioreactor. Apart from their ammonia removal activity, GS-HepG2 cells eliminated human tumor necrosis factor-α (TNF-α). Cytokine removal therefore promises to be another useful property of bioreactor cells.