Gie-Taek Chun

Mechanisms underlying TGF-β1-induced expression of VEGF and Flk-1 in mouse macrophages and their implications for angiogenesis

TGF‐β induces vascular endothelial growth factor (VEGF), a potent angiogenic factor, at the transcriptional and protein levels in mouse macrophages. VEGF secretion in response to TGF‐β1 is enhanced by hypoxia and by overexpression of Smad3/4 and hypoxia‐inducible factor‐1α/β (HIF‐1α/β). To examine the transcriptional regulation of VEGF by TGF‐β1, we constructed mouse reporters driven by the VEGF promoter. Overexpression of HIF‐1α/β or Smad3/4 caused a slight increase of VEGF promoter activity in the presence of TGF‐β1, whereas cotransfection of HIF‐1α/β and Smad3/4 had a marked effect. Smad2 was without effect on this promoter activity, whereas Smad7 markedly reduced it. Analysis of mutant promoters revealed that the one putative HIF‐1 and two Smad‐binding elements were critical for TGF‐β1‐induced VEGF promoter activity. The relevance of these elements was confirmed by chromatin immunoprecipitation assay. p300, which has histone acetyltransferase activity, augmented transcriptional activity in response to HIF‐1α/β and Smad3/4, and E1A, an inhibitor of p300, inhibited it. TGF‐β1 also increased the expression of fetal liver kinase‐1 (Flk‐1), a major VEGF receptor, and TGF‐β1 and VEGF stimulated pro‐matrix metalloproteinase 9 (MMP‐9) and active‐MMP‐9 expression, respectively. The results from the present study indicate that TGF‐β1 can activate mouse macrophages to express angiogenic mediators such as VEGF, MMP‐9, and Flk‐1.

Continuous Culture of Immobilized Streptomyces Cells for Kasugamycin Production

Continuous cultures of immobilized Streptomyces kasugaensis, a kasugamycin producer, were carried out on Celite beads. When using a prototype separator for immobilized‐cell separation and recycling, the continuous operation could not be sustained for an extended period as a result of an excessive loss of immobilized cells caused by the poor performance of the separator. Accordingly, the immobilized‐cell separator was revised to provide better immobilized‐cell settling and thus recycling into the reactor. In a subsequent culture using the revised separator, a stable operation was maintained for over 820 h with a high kasugamycin productivity. The kasugamycin productivity ranged from 9.8 to 16.1 mg/L/h, which was about 14‐ to 23‐fold higher than that in a batch suspended‐cell culture. When the original feeding medium concentration was doubled at the end of the continuous culture, the productivity became severely impaired for several reasons, which will be discussed. An excessive formation of free cells and loss of immobilized cells through the separator were also observed.

Enhancement of kasugamycin production by pH shock in batch cultures of Streptomyces kasugaensis.

Biosynthesis of kasugamycin could be greatly enhanced by applying a nonnutritional stress of pH shock, that is, sequential pH changes from a neutral pH to an acidic condition and then back to the neutral condition. During the acidic period, cell growth decreased to nil. After recovery of the neutral condition, the cell growth resumed after a time lag concurrently with the biosynthesis of kasugamycin at a greatly enhanced rate compared with the control case without a pH shock. In a series of experiments performed to identify the optimal length of pH shock, four different lengths (6, 12, 24, and 48 h) of pH shock were applied. The best result was obtained when pH shock was applied for 24 h, with kasugamycin productivity approximately 7‐fold higher than that of the control.

Ammonia removal using hepatoma cells in mammalian cell cultures

It was examined whether hepatocyte cell lines can be used for ammonia removal in mammalian cell cultures. It was found that there exists a critical ammonium concentration level for each hepatocyte cell to remove ammonia. Among the cells tested in this work, primary hepatocytes showed the strongest ammonia removal capability if ammonium concentration is higher than the critical level. However, primary hepatocytes lost the liver function gradually and finally died after 2−3 weeks. Because of this limitation, primary hepatocytes were not appropriate to be used for ammonia removal in long‐term cultures. Hep G2 cells, which are immortal, also showed a strong ammonia removal activity. The ammonia removal activity of Hep G2 cells depended on the concentration of ammonium in the medium, as in the case of primary hepatocytes. However, urea could not be detected in the course of ammonia removal by Hep G2 cells. Instead of urea, Hep G2 cells secreted glutamine into the culture medium. The capacity for ammonia removal was higher in the absence than in the presence of glutamine. Thus we checked the activity of glutamine synthetase in the Hep G2 cells. The level of glutamine synthetase activity increased with the addition of ammonium chloride. This result accounts for the ammonium concentration dependency of Hep G2 cells in ammonia removal and glutamine synthesis. Furthermore Hep G2 cells could grow well in the absence of glutamine, which was necessarily required in mammalian cell cultures. These results prove that glutamine formation serves as the primary mechanism of detoxifying ammonia in hepatocyte cell lines as expected. In addition, it was demonstrated that ammonium level could be reduced 38% and that erythropoietin production increased 2‐fold in the mixed culture of Hep G2 and recombinant CHO cells.

Characterization of a non-phosphotransferase system for cis,cis -muconic acid production in Corynebacterium glutamicum

Cis,cis-muconic acid (CCM) is a biochemical material that can be used for the production of various plastics and polymers and is particularly gaining attention as an adipic acid precursor for the synthesis of nylon-6,6. In the current study, the production of CCM was first attempted by introducing a newly developed protocatechuate (PCA) decarboxylase from Corynebacterium glutamicum 13032 to inha103, which completed the biosynthetic pathway therein. To improve CCM productivity, a phosphoenol pyruvate (PEP)-dependent phosphotransferase system (PTS) that consumed the existing glucose was developed, in the form of a strain with a non-PTS that did not consume PEP. To improve glucose uptake, we developed P25 strain, in which iolR (a transcriptional regulator gene) was additionally deleted. Strain P28, a P25 derivative expressing PCA decarboxylase, produced 4.01 g/L of CCM, which was 14% more than that produced by the parental strain. Moreover, strains P29 and P30, with an active pentose phosphate pathway and overexpressing important genes (qsuB) in the metabolic pathway, produced 4.36 and 4.5 g/L of CCM, respectively. Particularly, the yield per glucose in strain P30 was similar to that of the fed-batch culture of Escherichia coli, which has the highest reported yield of 22% (mol/mol). These results are underpinned by the characteristics of the non-PTS with increased PEP availability and a strain with deletion of the iolR gene, which greatly increased glucose uptake.

Development of Miniaturized Culture Systems for Large Screening of Mycelial Fungal Cells of Aspergillus terreus Producing Itaconic Acid

The task of improving a fungal strain is highly time-consuming due to the requirement of a large number of flasks in order to obtain a library with enough diversity. In addition, fermentations (particularly those for fungal cells) are typically performed in high-volume (100-250 ml) shake-flasks. In this study, for large and rapid screening of itaconic acid (IA) high-yielding mutants of Aspergillus terreus, a miniaturized culture method was developed using 12-well and 24-well microtiter plates (MTPs, working volume = 1-2 ml). These miniaturized MTP fermentations were successful, only when highly filamentous forms were induced in the growth cultures. Under these conditions, loose-pelleted morphologies of optimum sizes (less than 0.5 mm in diameter) were casually induced in the MTP production cultures, which turned out to be the prerequisite for the active IA biosynthesis by the mutated strains in the miniaturized fermentations. Another crucial factor for successful MTP fermentation was to supply an optimal amount of dissolved oxygen into the fermentation broth through increasing the agitation speed (240 rpm) and reducing the working volume (1 ml) of each 24-well microtiter plate. Notably, almost identical fermentation physiologies resulted in the 250 ml shake-flasks, as well as in the 12-well and 24-well MTP cultures conducted under the respective optimum conditions, as expressed in terms of the distribution of IA productivity of each mutant. These results reveal that MTP cultures could be considered as viable alternatives for the labor-intensive shake-flask fermentations even for filamentous fungal cells, leading to the rapid development of IA high-yield mutant strains.

Optimization of cultivation medium and fermentation parameters for lincomycin production by Streptomyces lincolnensis

Production of lincomycin by Streptomyces lincolnensis was studied by varying medium composition and environmental conditions. With the medium composition optimized by statistical experimental design (45 g/L soluble starch, 15 g/L sugar cane molasses, 13.33 g/L peptone water, 6.67 g/L NaNO3 and 4.0 g/L CaCO3), lincomycin production increased by 2 ∼ 3 fold compared to that obtained with un-optimized basal medium. Lincomycin production was further improved by optimizing culture conditions such as agitation speed, impeller type and pH under the optimized medium condition. The highest titer of 350 mg/L lincomycin was achieved from 240 h bioreactor culture. These results demonstrate that fermentation conditions for maximal lincomycin production by Streptomyces lincolnensis were optimized via biotechnological processes.

Repeated-batch culture of immobilizedGibberella fujikuroi B9 for gibberellic acid production: An optimization study

The performance of immobilized fungal cells on celite beads for the production of gibberrelic acid was investigated in flasks and 7-L stirred-tank reactor. Repeated incubations of immobilized fungal cells increased cell concentrations and volumetric productivity. The maximum volumetric productivity obtained in the immobilized-cell culture was 3-fold greater than that in suspended-cell culture. The concentration of cotton seed flour (CSF), amont the various nutrients supplied, most significantly influenced productivity and operational stability. Notably, insoluble components in CSF were found to be essential for production. CSF at 6 g/L with 60 g/L glucose was found to be optimal for gibberellic acid production and stable operation by preventing excessive cell growth.

Development of a cell-loaded biosupport separator for continuous immobilized-cell perfusion culture

An efficient cell‐loaded biosupport separator of the decantor type was developed and applied for a continuous perfusion culture to produce cyclosporin A (CyA), in which fungal cells were immobilized on Celite beads. In the preliminary experiments employing highly viscous polymer (carboxymethyl cellulose) solutions, the decantor showed good separation performances at high solution viscosites and dilution rates. Two concentric cylindrical tubes installed inside the decantor turned out to play key roles in the efficient separation of the immobilized cells. By installing the decantor on an immobilized‐cell perfusion bioprocess system, a stable continuous operation was possible even at a high dilution rate for an extended period, leading to high productivities of free cells and CyA. Almost no immobilized biomass existed in the effluent stream from the bioreactor, demonstrating the effectiveness of the decantor system. It is noteworthy that we could obtain these results despite unfavorable fermentation conditions, i.e., reduced apparent density of cell‐loaded beads and increased drag force on the bead particles caused by overgrowth of cells on the bead surface, tubulence caused by large air bubbles, and the existence of a high density of suspended fungal cells (10 g/L) in the fermentation broth.

Four-Week Repeated Intravenous Dose Toxicity and Toxicokinetic Study of TS-DP2, a Novel Human Granulocyte Colony Stimulating Factor in Rats.

TS-DP2 is a recombinant human granulocyte colony stimulating factor (rhG-CSF) manufactured by TS Corporation. We conducted a four-week study of TS-DP2 (test article) in repeated intravenous doses in male and female Sprague-Dawley (SD) rats. Lenograstim was used as a reference article and was administered intravenously at a dose of 1000 μg/kg/day. Rats received TS-DP2 intravenously at doses of 250, 500, and 1000 μg/kg/day once daily for 4 weeks, and evaluated following a 2-week recovery period. Edema in the hind limbs and loss of mean body weight and body weight gain were observed in both the highest dose group of TS-DP2 and the lenograstim group in male rats. Fibro-osseous lesions were observed in the lenograstim group in both sexes, and at all groups of TS-DP2 in males, and at doses of TS-DP2 500 μg/kg/day and higher in females. The lesion was considered a toxicological change. Therefore, bone is the primary toxicological target of TS-DP2. The lowest observed adverse effect level (LOAEL) in males was 250 μg/kg/day, and no observed adverse effect level (NOAEL) in females was 250 μg/kg/day in this study. In the toxicokinetic study, the serum concentrations of G-CSF were maintained until 8 hr after administration. The systemic exposures (AUC0-24h and C0) were not markedly different between male and female rats, between the administration periods, or between TS-DP2 and lenograstim. In conclusion, TS-DP2 shows toxicological similarity to lenograstim over 4-weeks of repeated doses in rats.