Biomedical subjects

Takeshi Omasa

Publications and source records attributed to Takeshi Omasa.

9 recordsLinked to original sources

PubMed2006

Utilization of hydrophobic bacterium Rhodococcus opacus B-4 as whole-cell catalyst in anhydrous organic solvents.

Rhodococcus opacus strain B-4, which has recently been isolated as an organic solvent-tolerant bacterium, has a high hydrophobicity and exhibits a high affinity for hydrocarbons. This bacterium was able to survive for at least 5 days in organic solvents, including n-tetradecane, oleyl alcohol, and bis(2-ethylhexyl) phthalate (BEHP), which contained water less than 1% (w/v). The biocatalytic ability of R. opacus B-4 was demonstrated in the essentially nonaqueous BEHP using indigo production from indole as a model conversion. By the catabolism of oleic acid for NADH regeneration, indigo production increased up to 71.6 microg ml(-1) by 24 h.

Alkanes↗

PubMed2006

Recognition of culture state using two-dimensional gel electrophoresis with an artificial neural network.

Proteomic technologies were applied to the examination of nutrient components in culture broth. In bioprocesses, many types of media have been proposed and used on the commercial scale. Natural nutrients, the chemical components of which cannot be identified completely, are often used in fermentation processes such as in the production of baker's yeast, alcoholic beverages, amino acids, and pharmaceuticals. The catabolic activities of the microorganisms in these processes vary with the species used. We used an artificial neural network (ANN) to recognize the sufficiency of chemical elements based on the protein spots resolved in 2-DE, and we evaluated this technique using the leave-one-out method. We also attempted to reduce the number of input data for spot selection based on sensitivity analysis of the ANN, and the selected data were used to improve accuracy.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2006

N-linked glycan structures of a mouse monoclonal antibody produced from tobacco BY2 suspension-cultured cells.

cDNA encoding H- and L-chains from a mouse monoclonal antibody was introduced into tobacco BY2 cells, and the resulting sugar chain structures of plant-produced antibodies were analyzed by a combination of HPLC, exoglucosidase digestion and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The glycan structures determined were Man(5-6)GlcNAc2 (22.3%), GlcNAcMan5GlcNAc2 (3.1%), GlcNAcMan3FucXylGlcNAc2 (24.4%), GlcNAcMan3XylGlcNAc2 (17.8%), Man3FucXylGlcNAc2 (24.3%), and Man3XylGlcNAc2 (8.1%). The major glycan structures of the antibodies produced by transgenic suspension-cultured cells contain typical plant bisecting beta(1,2)-xylose and alpha(1,3)-fucose residues, suggesting the posttranslational modification of a recombinant antibody in the late Golgi apparatus.

Animals↗

PubMed2005

The bioreactor with CYP3A4- and glutamine synthetase-introduced HepG2 cells: treatment of hepatic failure dog with diazepam overdosage.

A novel recombinant human hepatic cell line, CYP3A4- and glutamine synthetase (GS, an enzyme which converts ammonium ion and glutamate to glutamine)-introduced HepG2 (HepG2-GS-CYP3A4), was established. Its usefulness in a large-scale culture with a circulatory bioreactor in vitro and in dog models of ischemic hepatic failure with acute diazepam (DZP, a substrate of CYP3A4) overdosage was further examined. HepG2-GS-CYP3A4 expressed about 9 times larger amounts of CYP3A4 protein than a control. After incubation with HepG2-GS-3A4 cells in a circulatory bioreactor for 24 h, ammonia and DZP concentrations in the culture medium significantly decreased by about 40%. Furthermore, this system improved the survival time and decreased serum concentrations of DZP, ammonia, and transaminase in dogs with ischemic hepatic failure plus acute DZP overdosage. The mean survival time with bioreactor with HepG2-GS-3A4 was 42.7 +/- 3.6 h, which was significantly longer than that without reactor, with reactor (no cells), and with HepG2-GS (23.4 +/- 2.8, 22.1 +/- 2.4, and 31 +/- 3.7 h, respectively). Therefore, it is concluded that this bioartificial liver could be a good tool for the treatment of dogs with hepatic failure and that it could potentially be a bridging procedure to liver transplantation.

Animals↗

PubMed2005

Effect of sulfur sources on specific desulfurization activity of Rhodococcus erythropolis KA2-5-1 in exponential fed-batch culture.

The effects of sulfur sources on the desulfurization activity of Rhodococcus erythropolis KA2-5-1 were investigated by using an exponential fed-batch culture technique. The feed rate of a sulfur source was controlled independently of the feed rate of ethanol, which was used as a carbon and energy source. Among the sulfur sources examined were dibenzothiophene (DBT), ammonium sulfate, L-cysteine, L-methionine, and 2-amino-ethanesulfonic acid. When the fed-medium contained DBT as the sole sulfur source, KA2-5-1 cells showed a maximum desulfurization activity of approximately 130 mmol 2-HBP kg-cell(-1) h(-1). Similar levels of enzyme activity were also achieved with inexpensive ammonium sulfate by using the exponential fed-batch culture technique. In addition, higher levels of desulfurization activity were achieved by increasing the dosage of the DBT desulfurization (dsz) operon and dszD gene in R. erythropolis KA2-5-1. The recombinant strain showed a maximum desulfurization activity of approximately 250 mmol 2-HBP kg-cell(-1) h(-1) in the exponential fed-batch cultures.

Biodegradation, Environmental↗

PubMed2002

Gene amplification and its application in cell and tissue engineering.

Gene amplification means the repeated replication of a certain gene without a proportional increase in the copy number of other genes and is a widespread phenomenon in eukaryotes. It is an important developmental and evolutionary process in many organisms. This article focuses on mammalian gene amplification and its application in cell and tissue engineering. The dhfr gene amplification in Chinese hamster ovary (CHO) cells, the gene amplification mechanism, the selection protocol and the application of gene amplification were reviewed.

Journal Article↗

PubMed2002

Artificial mimicking of physiological active transport by a membrane co-cultured with two different cells: hepatic origin HepG2 and renal origin PCTL-MDR.

We designed a membrane culture unit on which 2 different cell lines were co-cultured to achieve selective and active transport of toxins. Hepatic origin HepG2 and renal origin multidrug-resistant gene-transduced proximal convoluted tubular cell line (PCTL-MDR) were cultured on the opposite sides of an expanded polytetrafluoroethylene membrane. The activity of testosterone hydroxylation by original HepG2 was very low; however, the cytochrome p450 (CYP) 3A4-transduced recombinant HepG2 metabolized the substrate efficiently. Testosterone added into the outer medium was hydrolyzed by HepG2, and the metabolites were preferentially transported to the inner medium by PCTL-MDR. [3H]-digoxin and [14C]-inulin were added to the outer medium; the digoxin was transported from the outer to inner space through the cell monolayer but the inulin was not, suggesting that the membrane actively transported only the substrate of the channel protein, MDR. The cells were irradiated (10 Gy) to prevent a membrane leak due to overgrowth. The irradiation did not induce apoptosis but resulted in long-lasting membrane function without leakage. The membrane co-cultured with hepatic and renal origin cells will enable a novel hemofiltration system with selective and active transport activities.

Animals↗

PubMed2002

Molecular cloning and sequencing of the human heme-regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase from bone marrow culture.

The cDNA encoding human heme-regulated eukaryotic initiation factor-2 alpha (eIF-2 alpha) kinase was cloned from a human bone marrow culture. Its deduced amino acid sequence comprised of 629 amino acids with a calculated molecular weight of 71,031 Da. RT-PCR analysis revealed that the gene was also expressed in heart, kidney, spleen, muscle, and stomach.

Amino Acid Sequence↗

PubMedDate not supplied

Construction and evaluation of drug-metabolizing cell line for bioartificial liver support system.

Focusing on drug metabolism in liver, we constructed and evaluated a drug-metabolizing bioartificial liver (BAL) support system. In a previous study, we constructed ammonia-metabolizing CHO and hepatoma-derived HepG2 cell lines by recombination of the glutamine synthetase (GS) gene. For further mimicking of liver metabolism, the human hepatoma-derived cell line HepG2 was transformed by the pBudCE-GS-CYP3A4 vector, which contains GS and drug-metabolizing CYP 3A4 genes. The constructed GS-3A4-HepG2 cell line showed 3A4 activity higher than that of human primary hepatocytes. The drug-metabolizing activity of BAL (BAL clearance) was evaluated using this cell line. The estimated clearance was higher than that of the human hepatocyte system.

Ammonia↗