Showing papers by "Toshiomi Yoshida published in 1999"

Stable expression of human β1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns

Abstract: β1,4-Galactosyltransferase (UDP galactose: β-N-acetylglucosaminide: β1,4-galactosyltransferase; EC 2.4.1.22) catalyzes the transfer of galactose from UDP-Gal to N-acetylglucosamine in the penultimate stages of the terminal glycosylation of N-linked complex oligosaccharides in mammalian cells. Tobacco BY2 cells lack this Golgi enzyme. To determine to what extent the production of a mammalian glycosyltransferase can alter the glycosylation pathway of plant cells, tobacco BY2 suspension-cultured cells were stably transformed with the full-length human galactosyltransferase gene placed under the control of the cauliflower mosaic virus 35S promoter. The expression was confirmed by assaying enzymatic activity as well as by Southern and Western blotting. The transformant with the highest level of enzymatic activity has glycans with galactose residues at the terminal nonreducing ends, indicating the successful modification of the plant cell N-glycosylation pathway. Analysis of the oligosaccharide structures shows that the galactosylated N-glycans account for 47.3% of the total sugar chains. In addition, the absence of the dominant xylosidated- and fucosylated-type sugar chains confirms that the transformed cells can be used to produce glycoproteins without the highly immunogenic glycans typically found in plants. These results demonstrate the synthesis in plants of N-linked glycans with modified and defined sugar chain structures similar to mammalian glycoproteins.

Two Distinct Mechanisms Cause Heterogeneity of 16S rRNA

Abstract: To investigate the frequency of heterogeneity among the multiple 16S rRNA genes within a single microorganism, we determined directly the 120-bp nucleotide sequences containing the hypervariable α region of the 16S rRNA gene from 475 Streptomyces strains. Display of the direct sequencing patterns revealed the existence of 136 heterogeneous loci among a total of 33 strains. The heterogeneous loci were detected only in the stem region designated helix 10. All of the substitutions conserved the relevant secondary structure. The 33 strains were divided into two groups: one group, including 22 strains, had less than two heterogeneous bases; the other group, including 11 strains, had five or more heterogeneous bases. The two groups were different in their combinations of heterogeneous bases. The former mainly contained transitional substitutions, and the latter was mainly composed of transversional substitutions, suggesting that at least two mechanisms, possibly misincorporation during DNA replication and horizontal gene transfer, cause rRNA heterogeneity.

Structures of N-Linked Oligosaccharides of Glycoproteins from Tobacco BY2 Suspension Cultured Cells

Abstract: The structures of N-linked sugar chains of glycoproteins expressed in tobacco BY2 cultured cells are reported. Five pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the glycoproteins by reversed-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were identified by two-dimensional PA-sugar chain mapping, ion-spray MS/MS analysis, and exoglycosidase digestions. The five structures fell into two categories; the major class (92.5% as molar ratio) was a xylose containing-type (Man3Fuc1Xyl1GlcNAc2 (41.0%), GlcNAc2Man3Fuc1Xyl1GlcNAc2 (26.5%), GlcNAc1Man3Fuc1Xyl1GlcNAc2 (21.7%), Man3Xyl1GlcNAc2 (3.3%)), and the minor class was a high-mannose type (Man5GlcNAc2 (7.5%)). This is the first report to show that α(1→3) fucosylation of N-glycans does occur but β(1→4) galactosylation of the sugar chains does not in the tobacco cultured cells.

Enhanced monoclonal antibody production by gradual increase of osmotic pressure

Abstract: The time length required for the adaptation of AFP-27 hybridoma cells to high osmotic pressure and the effect of a gradual increase of osmotic pressure on monoclonal antibody production were investigated. When the cells were subjected to an increase of osmotic pressure from 300 mOsmol kg-1 to 366 mOsmol kg- 1, the intracellular content of osmoprotective free amino acids reached a maximum level 6 h after the osmotic pressure was increased to 366 mOsmol kg-1. The same time period of 6 h incubation at 366 mOsmol kg-1 was required to obtain a high growth rate of AFP-27 cells at 440 mOsmol kg-1 when the cells were subjected to a two-step increase of osmotic pressure from 300 mOsmol kg-1 to 366 mOsmol kg-1 and then to 440 mOsmol kg-1. The time length for the physiological adaptation of the cells to 366 mOsmol kg-1 was consequently estimated to be 6 h. Osmotic pressure during batch cultivation was gradually increased from 300 mOsmol kg-1 to 400 mOsmol kg-1 with an adaptation time of at least 6 h. The specific growth rates following a gradual increase of osmotic pressure were higher than those at a constant osmotic pressure of 400 mOsmol kg-1, while the specific monoclonal antibody production rate increased with the increase in the mean osmotic pressure. As a result, the cells grown under a gradual increase of osmotic pressure produced higher amounts of monoclonal antibodies than did those grown under constant osmotic pressure.

Spatial development of the cultivation of a bone marrow stromal cell line in porous carriers

Abstract: The spatial development of the cultivation of a bone marrow stromal cell line (SR-4987) in porous carriers was investigated in order to construct a three-dimensional hematopoietic culture system. Low-rate continuous agitation, 20 rpm, was an appropriate method to achieve initial adhesion of cells onto a cellulose porous beads (CPB, 100 μm pore diameter) in a spinner bottle, compared with other methods such as centrifugation and intermittent agitation. Cell growth with continuous agitation at 70 rpm after initial cell adhesion was not inferior to that at 20 rpm. A 2- and 10-fold increase in the inoculum cell concentration for CPB and another type of porous cellulose beads (Micro-cube (MC), 500 μm pore diameter) resulted in a 1.2- and 2-fold increase in final cell concentrationm, respectively. Cells attached to the MC beads and a polyester nonwoven dic (Fibra-cell (FC)) could grow and spread well on the carriers and a fibroblast-like shape was observed under scanning electron microscopy while the cells on CPB were globular. The flatness and inner surface area of these carriers may be the reason for the differences in cell morphology.

Fluid mixing and oxygen transfer in cell suspensions of Taxus chinensis in a novel stirred bioreactor

Abstract: In high-density plant cell cultures, mixing and mass transfer are two key issues, which should be emphasized for process optimization. In this work, both mixing and oxygen transfer characteristics of cell suspensions ofTaxus chinensis were studied in a new centrifugal impeller bioreactor with a working volume of 1.2 L. The mixing time (tM) and the volumetric oxygen transfer coefficient (KLa) under different operational conditions were determined in both tap water and cell suspensions of 100–400 g fresh weight/L (i.e., 5.65–23.1 g DW/L). At an aeration rate of 0.1 L/min,tM decreased from 10.6s at 30 rpm to 2.89 s at 200 rpm under 100 g FW/L, and from 9.63 s (120 rpm) to 4.05 s (300 rpm) under 400 g FW/L. Compared with the effect of agitation, aeration was less significant to the suspension mixing. At a relatively high agitation speed (e.g., 200 rpm),tM remained almost the same even though aeration rate was changed from 0.1 to 0.4 L/min. ThetM value increased slowly from 3.98 to 5.26 s at 120 rpm when the cell density was raised from 100 to 250 g FW/L. A rapid increase of bothtM and the suspension viscosity was observed at a cell density above 300 g FW/L. As expected, theKLa value increased with an increase of aeration rate and agitation speed, but decreased with an increase of cell density. The quantitative data obtained here are useful to investigate the effect of mixing stress on the cell physiology and metabolism ofTaxus chinensis in the bioreactor.

Beta-n-acetylgalactosaminidase gene

Abstract: PROBLEM TO BE SOLVED: To obtain the subject gene for producing β-N-acetylgalactosaminidase having high specificity to N-acetyl-β-galactosaminidase bond, and useful for elucidation of the structure of a sugar chain and biological activities thereof, by a genetic engineering technique. SOLUTION: The subject gene is an isolated DNA including a nucleotide sequence encoding a polypeptide capable of separating N-acetyl-D galactosamine present in the nonreducing terminal of a sugar chain and bound by β-glucoside bond but not separating N acetyl D glucosamine at the nonreducing terminal and bound by the β-glucoside bond, and having β-N-acetylgalactosaminidase activities (or functionally same activities as them). The gene is obtained by culturing Bacillus sp. AT 173-1 (FERM P-15390) strain, isolating the β-N- acetylgalactosaminidase from the culturing liquid, determining the N-terminal amino acid sequence of the separated β-N-acetylgalactosaminidase, synthesizing a primer, etc., for a PCR method, and cloning the subject gene.

Computer application in bio-process engineering in Japan

Abstract: The recent development of the researches conducted in Japan about computer applications in bio-process engineering is summarized, specially focusing on the application of knowledge engineering approaches for bio-process control. The topics on the methodological investigation, their applications for process development, and examples of industrial applications of expert systems, artificial neural networks, fuzzy theory, and genetic algorithm are thoroughly reviewed, and the contents of some representative recent works are introduced more extensively.

Procede de production de glycoproteines a glycosilation de type humain

Abstract: La presente invention concerne un procede de production d'une glycoproteine ayant une chaine de sucre du type humain comprenant une etape dans laquelle une cellule vegetale transformee est obtenue par introduction dans une cellule vegetale du gene de la glycosiltransferase et du gene d'une glycoproteine exogene, ainsi qu'une etape dans laquelle la cellule vegetale obtenue transformee est mise en culture.