Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells.

In order to get the high liquefaction yield from marine algae cell mass to fuel oil, the effect of salt stress on the accumulation of lipids and triacylglyceride in Dunaliella cells was investigated. Although initial NaCl concentration higher than 1.5 M markedly inhibited cell growth, increase of initial NaCl concentration from 0.5 (equal to sea water) to 1.0 M resulted in a higher intracellular lipid content (67%) in comparison with 60% for the salt concentration of 0.5 M. Addition of 0.5 or 1.0 M NaCl at mid-log phase or the end of log phase during cultivation with initial NaCl concentration of 1.0 M further increased the lipid content (70%).

Effect of chondroitin sulfate and hyaluronic acid on gene expression in a three-dimensional culture of chondrocytes.

The effect of glycosaminoglycan addition on a three-dimensional (3D) culture of porcine chondrocyte cells was investigated with a view to use in cartilage regenerative medicine. Chondroitin sulfate C increased the mRNA expression of type 2 collagen, while chondroitin sulfate A did not. Hyaluronic acid of high molecular weight markedly decreased the mRNA expression of both aggrecan and type 2 collagen, although hyaluronic acid of low molecular weight showed no apparent effect.

Adsorption of Lithocholic Acid to Fusarium equiseti M41 as an Essential Process in Its Conversion to Ursodeoxycholic Acid.

Fusarium equiseti M41 converts lithocholic acid to ursodeoxycholic acid. Adsorption of lithocholic acid particles to mycelia of F. equiseti M41 is essential in the conversion of lithocholic acid to ursodeoxycholic acid. Production of ursodeoxycholic acid was negligible when particles of lithocholic acid were absent. As the concentration of lithocholic acid particles increased, both the amount of mycelium-bound lithocholic acid and the production of ursodeoxycholic acid increased hyperbolically (K(1/2) = 1.9 g/liter and K(m(apparent) ) = 1.9 g/liter. A fluorescent lithocholic acid derivative was used to confirm that insoluble particles of lithocholic acid attached to the surface of the mycelia. The hydrophobic nature of this binding was estimated from the close relationship observed between the hydrophobicity of bile acids and their binding capacity to the mycelia. By repeated washing with 30% dimethyl sulfoxide, two binding modes of lithocholic acid were distinguished, i.e., surface binding (59% of bound lithocholic acid) and tight binding (41% of bound lithocholic acid). From the amount of tightly bound lithocholic acid, the intracellular concentration of lithocholic acid was calculated to be 1,433-fold higher than its saturating concentration in the reaction mixture, thus promoting effective conversion to ursodeoxycholic acid in the mycelia. Several lines of evidence indicated that glycoproteins of the cell wall participated in the binding of lithocholic acid.

Expanded bed adsorption for purification of alcohol dehydrogenase using a dye-iminodiacetic acid matrix.

Expanded bed adsorption (EBA) was successfully applied for the purification of alcohol dehydrogenase (ADH) from unclarified bakers' yeast homogenate using a new dye-iminodiacetic acid (IDA) matrix carrying immobilized metal ions. The existence of cells and cell debris do not have a significant effect on the adsorption of ADH on the matrix. The presence of imidazole in adsorption buffer was found to be an important factor in reducing the level of the adsorbed contaminant proteins. The optimal imidazole concentration was 5 mM. The dynamic binding capacity of the dye-IDA matrix in EBA was found to be 569 U/ml matrix. A chromatography of one-step elution with imidazole was developed, in which ADH was successfully adsorbed in the presence of 5 mM imidazole; subsequently, the column was washed with 5 mM imidazole containing 1 M NaCl and eluted with 150 mM imidazole containing 1.5 M NaCl. The purification factor and ADH recovery were 8.8 and 93.5%, respectively.

In vitro proliferation of primary rat hepatocytes expressing ureogenesis activity by coculture with STO cells.

A coculture system for in vitro proliferation of rat primary hepatocytes with feeder cells was investigated with the view of constructing a hybrid artificial liver module using human hepatocytes. A mouse cell line, STO, was apparently effective compared with other kinds of feeder cells such as FLS5 and MRC5 in increasing the total cell number in the coculture of rat primary hepatocytes. The parenchymal hepatocyte, which are polygonal cells, spread well as the cultivation progressed. Monitoring of parenchymal hepatocyte colonies under a microscope showed that the area of each colony increased as the single culture and cocultures progressed. The adhesion area per hepatocyte increased little during the coculture with STO cells, while the adhesion area increased markedly in the single culture of hepatocytes. The density of hepatocytes increased more than two times during the coculture for 5 d, while it decreased during the single culture. The production rate of urea by hepatocytes markedly increased during the coculture, while the rate in the single culture was lower than the coculture and increased only slightly. The specific rate of urea production by hepatocytes in the coculture did not decrease even as the hepatocytes grew. Consequently, rat primary hepatocytes could proliferate in vitro by coculture with STO cells without a decrease in urea production activity.

Effect of concentrations of murine stromal and hematopoietic cells on the progenitors expansion in their three-dimensional coculture in nonwoven fabrics.

The effects of concentrations of murine stromal and hematopoietic cells on their three-dimensional coculture in nonwoven fabrics were investigated for the construction of an ex vivo expansion system for hematopoietic progenitors without adding expensive cytokines. The increase in initial stromal cell concentration from 0.4 x 10(5) to 2.7 x 10(5) cells/ml increased progenitor concentration, while the increase in initial hematopoietic cell concentration from 0.6 x 10(5) to 6.1 x 10(5) cells/ml decreased the fold increase in hematopoietic cell concentration.

Combined addition of glutathione and iron chelators for decrease of intracellular level of reactive oxygen species and death of Chinese hamster ovary cells.

The combined addition of reduced form of glutathione (GSH) and iron chelators for decrease of the intracellular level of reactive oxygen species (ROSs) and death of Chinese hamster ovary (CHO) cells was investigated. The addition of GSH to the serum-free cultures of CHO cells markedly lowered the intracellular ROS level, which effect was comparable to that of the addition of serum (0.4, 10%) and might be due to the reduction of ROS. The addition of iron chelators, deferoxamine and aurintricarboxylic acid, to the serum-free cultures also decreased the intracellular ROS level and increased the residual concentration of viable cells during the serum-free maintenance culture, which should be due to the inhibition of the Fenton and Harber-Weiss reactions stimulating the synthesis of ROS. The combined addition of GSH and the chelators decreased ROS level and cell death more effectively compared with their single addition.

Repeated addition of insulin for dynamic control of apoptosis in serum-free culture of Chinese hamster ovary cells.

Repeated addition of insulin, together with an initial addition of the reduced form of glutathione (GSH) and a chelator, aurintricarboxylic acid (ATA), was proposed as a strategy for the dynamic control of apoptosis in serum-free culture of Chinese hamster ovary (CHO) cells. Insulin suppressed cell death in serum-free culture by means of lowering intracellular reactive oxygen species levels via an increase in the specific glucose consumption rate. This effect of insulin addition was found not only at 0 h but also at 48 h during serum-free cultivation. In consideration of the reduction of insulin by GSH, insulin was again added at 24 h following the initial addition during the serum-free cultivation. The repeated addition of insulin decreased cell death and increased the production of tissue plasminogen activator compared with the single addition at 0 h.

Transduction of static pressure signal to expression of human granurocytes macrophage colony stimulating factor mRNA in Chinese hamster ovary cells.

The expression level of human granurocytes macrophage colony stimulating factor (hGM-CSF) mRNA in Chinese hamster ovary (CHO) DR1000L4N cells increased by 24% with pressurization. Treatment of cells with chelerythrine chloride (10 nM, 15 min), an inhibitor of protein kinase C (PKC), did not suppress the pressure-induced (0.9 MPa) expression of hGM-CSF mRNA, while it decreased the hGM-CSF gene expression level at normal pressure (0.1 MPa). Treatment with U0126 (20 microM, 60 min), a specific inhibitor of extracellular signal-related kinase (ERK1/2), decreased the expression level of hGM-CSF mRNA at 0.1 MPa to 80% of that without U0126 treatment. Similarly, treatment with U0126 decreased the pressure-induced (0.9 MPa) expression level of hGM-CSF mRNA to 79% of the control expression level at 0.1 MPa without treatment. The amount of intracellular phosphorylated ERK1/2 increased with pressurization (0.9 MPa). These results suggest that the pressure-induced expression of hGM-CSF mRNA in CHO DR1000L4N cells depends not on PKC but on the ERK1/2 signaling cascade.

Enhanced glutamic acid production of Brevibacterium sp. with temperature shift-up cultivation.

For cells of Brevibacterium sp. under conditions of biotin limitation, the efflux of metabolites through the cell membrane was affected by temperature. After the temperature shift-up from 30 degrees C to 38 degrees C, both the specific production rate of glutamic acid and the excretion of intracellular materials, such as glucose-6-phosphate and nucleotides, were increased simultaneously. For the production of glutamic acid, not only the yield but also the specific production rate was increased by the temperature shift-up.

Effect of poly DL-lactic-co-glycolic acid mesh on a three-dimensional culture of chondrocytes.

Collagen gel and a copolymer mesh of polylactate and polyglucuronic acid (PLGA) were combined for a three-dimensional (3D) culture of chondrocyte cells having both uniform cell distribution and mechanical strength. Although the 3D culture in 96-multi-wells caused decreases in the glucose consumption rate and cell density in the latter stages of cultivation, transfer of the culture gel from a 96-multi-well plate to a 24-multi-well plate and an increase in medium volume effectively increased the glucose consumption rate and the accumulation of glycosaminoglycan (GAG) in the gel. The reason for the decrease in glucose consumption rate in a 96-multi-well plate was not the depletion of glucose or the accumulation of lactate in the gel, but the accumulation of degradation products of PLGA.

Development of new dye-metal agarose-coated alumina matrix and elution strategy for purification of alcohol dehydrogenase.

A high-density matrix was prepared by coating an alumina particle with agarose using an emulsion technique. Iminodiacetic acid and Cibacron Blue 3GA were immobilized onto this matrix. Charging this matrix with zinc created a useful chromatography matrix for purification of yeast alcohol dehydrogenase. The elution strategy was then investigated to obtain a high recovery of this enzyme and a high purification factor. One-step elution using 4 mM EDTA containing 0.5 M NaCl resulted in 66% enzyme recovery and a purification factor of 4.7. Two-step elution using imidazole containing NaCl resulted in a higher purification factor. The first-step elution using 5 mM imidazole containing 1 M NaCl released most contaminant proteins. The second-step elution using 150 mM imidazole containing 1.5 M NaCl resulted in high-performance purification with a purification factor of 6.5 and an enzyme recovery of 40.7%. Equilibration of the matrix with imidazole prior to sample application increased the purification factor and the enzyme recovery to 8.4 and 76.8%, respectively.

Analysis of hematopoietic microenvironment containing spatial development of stromal cells in nonwoven fabrics.

We previously reported that a 3D coculture system for hematopoietic cells with spatial development of stromal cells in nonwoven fabrics was superior to a conventional 2D coculture system with respect to progenitor expansion and stem cell maintenance. The hematopoietic environments in 2D and 3D coculture systems were compared and it was found there were no differences in the amounts of soluble factors, e.g., mRNA levels of stem cell factor (SCF) and thrombopoietin (TPO), the secretion of SCF, and hematopoietic stimulating activity of culture supernatant. On the other hand, the total amount of insoluble factors such as extracellular proteins around the stromal cells and the mRNA level of laminin alpha5 were apparently higher in the 3D culture.

Multivalent binding interaction of alcohol dehydrogenase on dye-metal affinity matrix.

The optimization of a chromatographic process using immobilized metal affinity chromatography requires an understanding of the factors that govern the interaction between proteins and immobilized metal ions. Factors, such as concentrations of protein, NaCl and imidazole were investigated to elucidate kinetics of adsorption of alcohol dehydrogenase (ADH) onto a dye-iminodiacetic acid matrix (dye-IDA matrix). The results indicate that the adsorption of ADH onto a dye-IDA matrix occurs in the mode of multiple-site binding interactions between ADH and zinc ions immobilized on the dye-IDA matrix. The estimated average number of interaction sites was 4.5 and the association constant was 6 x 10(-9) mM(-n). The isotherm of ADH adsorption was well represented by a multivalent model of protein-zinc ion interactions. For the adsorption of ADH from clarified yeast homogenate, addition of imidazole as a protein competitor to adsorption buffer increased the adsorption specificity of ADH, thereby suppressing contaminant protein adsorption. It was also observed that the adsorption of ADH was better performed at high initial protein concentrations in the yeast homogenate. Consequently, these results may have important implications on the optimization of the strategy for immobilized metal affinity adsorption in packed and expanded bed systems.

Effect of sugar residues in a glycolipid coated onto a dish on ammonia consumption and gluconeogenesis activity of primary rat hepatocytes.

Coating of 3,4,5-tris(dodecyloxy)benzyl-beta-D-galactopyranoside (TDOB-Gal) on a dish for suspension culture increased the specific ammonia consumption rate of primary rat hepatocytes to 2.4 times that in the case of rat hepatocytes cultured in a dish without coating while there was no increase in the specific urea production rate. TDOB-beta-D-glucopyranoside (TDOB-Glc), -alpha-D-mannnoside, -beta-D-mannoside, 2-acetamido-2-deoxy-beta-D-glucopyranoside, and 2-acetamido-2-deoxy-beta-D-galactopyranoside had almost no influence on the above-mentioned specific rates. In the ammonia loading assay, cells on the dish with TDOB-Gal and -Glc coatings produced glucose, suggesting gluconeogenesis.

Effect of static pressure on human granulocyte-macrophage stimulating factor (hGM-CSF) production by Chinese hamster ovary cells.

The effect of static pressure on the production of human granulocyte-macrophage colony stimulating factor (hGM-CSF) by the Chinese hamster ovary (CHO) cell line DR1000L4N was investigated. Although constant static pressures between 0.15 and 0.9 MPa had little influence on specific growth rate, the specific production rate of hGM-CSF increased from 1.23 to 1.62 x 10(-13) mg/cell/h in proportion to static pressure. The specific rate of tPA production by the human embryo lung cell line MRC-5 also increased from 2.92 to 6.38 x 10(-12) mg/cell/h as static pressure increased. The expression level of hGM-CSF mRNA increased with the pressurization even for 4 h.

Characterization of almond alpha-mannosidase and its application for structure analysis of sugar chain.

Almond alpha-mannosidase was purified by separation on columns of DEAE-Sephadex A50 and hydroxyapatite, and characterized. Its optimum pH was approximately 3.8. It was also shown to be stable from pH 6 to 8. Its activity was stable up to 60 degrees C. The thermostability of almond alpha-mannosidase at 73 degrees C appeared to be superior to that of jack bean a-mannosidase. We examined the substrate specificity of the former toward high-mannose-type N-glycan Man9GlcNAc2, and showed that the deduced trimming pathway was more diverse than that of the latter. We could use almond alpha-mannosidase as well as jack bean alpha-mannosidase for analysis of sugar chain structures.

Effect of mixing time on taxoid production using suspension cultures of Taxus chinensis in a centrifugal impeller bioreactor.

The effects of fluid mixing on the cell growth and secondary metabolite production of plant cells were investigated in a low-shear centrifugal impeller bioreactor (CIB) system. Suspension cultures of Taxus chinensis cells producing taxuyunnanine C (Tc), a physiologically active secondary metabolite, were used as a model system for this investigation. The mixing time (t(m)) and volumetric oxygen transfer coefficient (k(L)a) in the bioreactor were characterized at various cell densities and operating conditions. A constant t(m) of 5 s or 10 s was maintained during cultivation by adjusting the impeller agitation speed with no detrimental effect on the cultured cells. A higher cell density, Tc content and total Tc production were obtained under the shorter mixing time of 5 s. The favorable effect of more rapid mixing on Tc production was also confirmed when the Tc accumulation was significantly increased through culture elicitation using 100 microM methyl jasmonate (MJA). The lower Tc production at the longer t(m) of 10 s was mainly attributed to oxygen transfer limitation in the dead zones and larger cell aggregates resulting from poor mixing.

Differentiation of Human Bone Marrow Mesenchymal Stem Cells to Chondrocytes for Construction of Three-dimensional Cartilage Tissue.

A differentiation method of human bone marrow mesenchymal stem cells (MSCs) to chondrocytes was developed for the construction of a three-dimensional (3D) cartilage tissue. The adhesive cells, which were isolated from a human bone marrow aspirate were embedded in type I collagen in a poly-L: -lactate-glycolic acid copolymer (PLGA) mesh and cultivated for 4 week together with growth factors. The degree of cellular differentiation was estimated by quantitative RT-PCR of aggrecan and type II collagen mRNAs and by staining with Safranin O. The 3D culture showed a higher degree of differentiation even without growth factors than the conventional pellet culture with growth factors, namely, dexamethasone and transforming growth factor (TGF)-beta 3. The 3D culture for 2 week with the combined addition of dexamethasone, TGF-beta 3, and insulin-like growth factor (IGF)-I reached a 30% expression of aggrecan mRNA compared with that in primary human chondrocytes, while the aggrecan mRNA expression in the conventional pellet culture was less than 2%. The sequential two-step differentiation cultivation, during which the cells were cultivated in 3D for 1 week after the conventional two-dimensional (2D) culture for 1 week, could markedly accelerate the expression of aggrecan mRNA compared with the 3D cultivation for 2 week.

Effect of Subcultivation of Human Bone Marrow Mesenchymal Stem on their Capacities for Chondrogenesis, Supporting Hematopoiesis, and Telomea Length.

Effects of subcultivation of human bone marrow mesenchymal stem cells on their capacities for chondrogenesis and supporting hematopoiesis, and telomea length were investigated. Mesenchymal stem cells were isolated from human bone marrow aspirates and subcultivated several times at 37 degrees C under a 5% CO(2) atmosphere employing DMEM medium containing 10% FCS up to the 20th population doubling level (PDL). The ratio of CD45(-) CD105(+) cells among these cells slightly increased as PDL increased. However, there was no marked change in the chondrogenic capacity of these cells, which was confirmed by expression assay of aggrecan mRNA and Safranin O staining after pellet cell cultivation. The change in capacity to support hematopoiesis of cord blood cells was not observed among cells with various PDLs. On the other hand, telomere length markedly decreased as PDL increased at a higher rate than that at which telomere length of primary mesenchymal stem cells decreased as the age of donor increased.