Development of a fed-batch culture process for enhanced production of recombinant human antithrombin by Chinese hamster ovary cells

Antithrombin is a serine protease inhibitor that inactivates several coagulation proteases, primarily thrombin and factor Xa. The Chinese hamster ovary (CHO) cell line transfected with a vector expressing recombinant human antithrombin (rAT) and a selectable marker, glutamine synthetase (GS), was cultivated in a 2-l fed-batch culture process using serum-free, glutamine-free medium. To maximize the rAT yield, effects of culture pH, balanced amino acid feeding, and an increased glutamate concentration on cell metabolism and rAT production were investigated. When cells were grown at pH values of 6.6, 6.8, 7.0, and 7.2, the maximum cell density and maximum lactate concentration decreased with decreasing pH. The highest production level of rAT was obtained at culture pH 6.8 due to the extended culture lifetime. Compared to the imbalanced amino acid feeding at culture pH 6.8, the balanced amino acid feeding increased the amount of rAT activity by 30% as a result of an increased viable cell number. A decrease in the specific glucose consumption rate (q(Glc)) with increasing culture time was observed in all the above-mentioned experiments, while the glucose concentration was maintained above 0.7 g l(-1). In addition, a decrease in the specific rAT production rate (q(rAT)) was observed after the depletion of lactate in the late cultivation stage. Taken together, these results suggest that the reduced availability of cellular energy caused by the decrease in q(Glc) and depletion of lactate led to the decrease in q(rAT). This decrease in q(rAT) was partially prevented by increasing the residual glutamate concentration from 1 mM to 7 mM, thus resulting in an additional 30% increase in the amount of rAT activity. The optimized fed-batch culture process yielded 1.0 g l(-1) rAT at 287 h of cultivation.     

Novel flexible network polymers consisting of oligomeric primary polymer chains originated in the mechanistic discussion of multiallyl crosslinking polymerization

Summary It is well known that allyl monomers polymerize only with difficulty and yield polymers having low molecular weights, i.e., oligomers. Inevitably, free-radical multiallyl crosslinking polymerization provides network polymers consisting of oligomeric primary polymer chains, i.e., having abundant dangling chains. This led to the development of novel flexible network polymers such as amphiphilic network polymers (I) consisting of short primary polymer chains and long crosslink units with opposite polarities, simultaneous interpenetrating networks (II) consisting of both polyurethane (PU) and polymethacrylate (PM) networks with oligomeric primary polymer chains, and network polymers (III) consisting of centipede-type primary polymer chains. Thus, the solution copolymerizations of benzyl methacrylate with tricosaethylene glycol dimethacrylate in the presence of lauryl mercaptan yielded I consisting of nonpolar, short primary polymer chains and polar, long crosslink units. The opposite type of I was prepared by the copolymerization of 2-hydroxyethyl methacrylate, a polar monomer having a hydroxyl group, with heneicosapropylene glycol dimethacrylate, a nonpolar monomer having a poly(oxypropylene) unit. The equimolar polyaddition crosslinking reaction of poly(methyl methacrylate-co-2-methacryloyloxyethyl isocyanate) with tri(oxytetramethylene) glycol, leading to PU networks, and the free-radical crosslinking copolymerization of methyl methacrylate with tri(oxytetramethylene) dimethacrylate in the presence of CBr₄, leading to PM networks, were progressed simultaneously, providing II formed via the topological crosslink between PU and PM network structures. The post-copolymerizations of oligomeric allyl methacrylate/alkyl methacrylate precopolymers, having different amounts of pendant allyl groups and different molecular weights, with allyl benzoate/vinyl benzoate monomer mixtures were conducted to give III.     

Epitaxially grown free-standing diamond platelet

We obtained an epitaxially grown free-standing diamond platelet utilizing epitaxial diamond film formed on a {100} iridium surface using a d.c. plasma CVD process. Iridium was selected as a suitable substrate material for the heteroepitaxy of diamond based on original criteria. Confocal Raman spectroscopy revealed that the diamond platelet contained little or no non-diamond carbon. The obtained diamond platelet is transparent to visible light and cleavable along the 110 direction on the surface. The angles between the top surface and the cross-sectional surfaces are approximately 55°, almost equal to the theoretical angle of 54.74° between {100} and {111} planes in cubic crystals. Therefore, the cross-sectional surfaces would be {111} planes of a typical facet for single-crystalline diamond. This means that the diamond platelet we have formed has relatively good crystallinity.     

Temperature and Stress Distributions in a Hollow Cylinder of Functionally Graded Material: The Case of Temperature-Independent Material Properties

We have presented a numerical technique for analyzing one-dimensional transient temperature distributions in a circular hollow cylinder that was composed of functionally graded ceramic–metal-based materials, without considering the temperature-dependent material properties. The functionally graded material (FGM) cylinder was assumed to be initially in a steady state of gradient temperature; the ceramic inner surface was exposed to high temperature, and the metallic outer surface, which was associated with its in-service performance, was exposed to low temperature. Then, the FGM cylinder was cooled rapidly on the ceramic surface of the cylinder, using a cold medium. The transient temperature and related thermal stresses in the FGM cylinder were analyzed numerically for a model of the mullite–molybdenum FGM system. The technique for analyzing the temperature distribution is quite simple and widely applicable for various boundary conditions of FGMs, in comparison with methods that have been proposed recently by other researchers.     

Effect of the addition of NH4F on anodic behaviors of DSA-type electrodes in H2SO4-(NH4)2SO4 solutions

The effect of NH₄F addition on the high-anodic behaviors of various DSA-type electrodes was investigated in a mixture of sulfuric acid and ammonium sulfate. The pronounced effect of NH₄ addition was observed on DSA-type Ti/RuO₂, Ti/IrO₂?TiO₂ electrodes, but not observed on the other electrodes studied. The close relationship was found between the increment of the electrode potential caused by the NH₄ addition and the quantity of F^? adsorbed on the electrode surface. The effect of NH₄F addition was considered to be resulted from the adsorption of F^? on the electrode surface, and the presence of adsorbed F^? was directly proved.     

Development of novel method for screening microorganisms using symbiotic association between insect (Coptotermes formosanus Shiraki) and intestinal microorganisms

It is becoming increasingly difficult to isolate novel useful microorganisms from the natural environment using conventional screening methods based on pure culture techniques. A novel method for screening microorganisms in symbiotic association with insects was developed. This method involves the following two steps. In the first step, the existence of desired microorganisms that grow well by degrading difficult-to-degrade materials in the gut of insects is detected using the survivability of insects as an indicator. In the second step, the desired microorganisms are selected from the surviving insects. The second step is based on an idea that the guts of insects act as continuous-culture systems whereby microorganisms that cannot degrade diet components are washed out whereas those that can degrade diet components are retained and made to multiply in the gut. Coptotermes formosanus Shiraki was fed with an artificial diet containing phenol as a model of lignin-derived and difficult-to-degrade compound. Each C. formosanus feeding on an artificial diet containing 100 mg/l phenol had different levels of adaptation to the toxicity of phenol. About 20% of C. formosanus fed with an artificial diet containing 100 mg/l phenol died within a few days whereas others survived for more than 10 d. The structure of the intestinal microorganisms of the surviving C. formosanus fed with the 100 mg/l phenol artificial diet gradually changed and was very different from that of the bacterial communities obtained from the enrichment culture of wood-feeding C. formosanus using an artificial medium containing phenol as a sole carbon source. Furthermore, Only three species (as DGGE band) were detected from the gut of wood-feeding C. formosanus, whereas 200 times more phenol-degrading microorganisms were detected in the gut of C. formosanus feeding on a phenol artificial diet. Out of these nine species (as DGGE band) of phenol-degrading microorganisms were isolated. The screening method developed in this study can also be applied to various insects, leading to the isolation of various microorganisms that can degrade difficult-to-degrade compounds.     

Extraction of lanthanide ions from aqueous solution by bis(2-ethylhexyl)phosphoric acid with room-temperature ionic liquids

Extractions of five kinds of lanthanide metal ions by bis(2-ethylhexyl)phosphoric acid (DEHPA) with [1-Cn-3-methylimidazolium][PF₆](Cn = C₂, C₄) or [1-butyl-4-methylpyridinium][PF₆] were carried out under various DEHPA and HNO₃ concentrations from 0 to 1 M and under different temperature conditions from 298 to 333 K. These results were compared with those using the conventional organic solvent, hexane, in terms of their distribution coefficient values. Under all of the conditions in this study, the ionic liquid system shows more than three times greater extractability for lanthanide compared to when hexane was used. The distribution coefficient of lanthanide ions decreased as the length of the alkyl chain increased from the ethyl to the butyl. In addition, the imidazolium cation generally shows a higher distribution coefficient compared to the pyridinium cation in an ionic liquid. The concentration ratio of lanthanides and DEHPA resulted in an extraction affinity transition for lanthanides. Also evaluated in this study were issues related to the selectivity associated with the lanthanide mixture and the dependency of the ionic radius during lanthanide extraction.     

Relative reactivity of phenylboronic acid to phenylboronate ion toward L-lactate and mandelate at physiological pH

Kinetic studies were performed on the reactions of phenylboronic acid with L-lactic acid and mandelic acid in acidic aqueous and alkaline solutions in order to specify reactive species in these reactions. It was confirmed that the diprotonated ligand (H₂L: L-lactic acid or mandelic acid) is less reactive than the monoprotonated ligand (HL^?: L-lactate ion or mandelate ion), which made possible direct determination of the rate constants of phenylboronic acid (PhB(OH)₂) and its conjugate base, phenylboronate ion (PhB(OH)₃^?). It was found that PhB(OH)₂ is more reactive than PhB(OH)₃^?. On the basis of kinetic results, it was concluded that the most reactive species are PhB(OH)₂ and HL^? at physiological pH 7.4, so the reaction in the boronic acid-based sensor for L-lactate mainly would occur between these species.     

Heterogeneous nucleation of organic vapours on a cold substrate

The heterogeneous phase transition of the organic vapours, cyclohexane and p-xylene, on a cold substrate was observed by cooling the substrate at a slow rate under reduced pressure conditions. Either organic vapour at a vapour pressure lower than 130 Pa formed directly solid crystals but p-xylene vapour at a higher vapour pressure formed liquid condensates with subsequent formation of solid crystals. The dependence of the critical supersaturation ratios on the critical temperatures and the physical properties of the organic vapours has been clarified by the equation derived on the basis of classical nucleation theory.