Earticle

Immunosensor for the determination of LDL(Low-Density Lipoprotein), a good indicator for the diagnosis of atherosclerosis and hypercholesterolemia, was developed by using quartz crystal microbalance(QCM). The immunosensor consists of flow-through cell, oscillating circuit, oscilloscope, and frequency counter. FIA(Flow Injection Analysis) was applied to the QCM system for the measurement of LDL in liquid phase. Antibody showing binding affinity against LDL was immobilized on the gold electrode of a quartz crystal by covalent coupling via polyethylenimine / glutaredehyde. LDL was injected and bound to the antibody immobilized on the QCM immunosensor. The response of the immunosensor (F0 - F1) was found to be proportional to the LDL concentration from 200ug /ml to 800ug /ml. Operational conditions for the operation of immunosensor were also investigated in terms of sensitivity and non-specific binding.

About 75 strains which utilize cholesterol as sole carbon and energy source were isolated from 10 samples of Kimchi and 18 samples of fermented fish food (2 Ojingo-jeots, salt-fermented squid ; 5 Changran-jeots, salt-fermented pollack tripe ; 5 Myungran-jeots, salt-fermented Alaska pollack roe ; 3 Gajami-sikhae-jeots, fermented flat fish ; 2 Gul-jeots, salt-fermented oyster ; a Juneo-jeots, salt-fermented shad). Among them tested, the 3T6-5Mj strain isolated from Changran-jeot showed the highest activity on cholesterol degradation. The optimal composition of medium for the producing cholesterol degradation enzyme by 3T6-5Mj strain was 1.0 g/L NH4NO3, 1.0 g/L K2HPO4, 0.1 g/L MgSO4.7H2O, 1.0 g/L FeSO4.7H2O, 1 g/L NaCl, 5 g/L Trypton, 1 g/L Cholesterol, and 5 g/L Maltose at 30℃, pH 7.5, and the enzyme production reached a maximum level at 140 hours of cultivation

Addition of cyclodextrin in the biotransformation of digitoxin into digoxin by Digitalis lanata cell suspension cultures enhanced the conversion yield. Presence of cyclodextrin also supported good stability of the intermediate product, digoxin, for long time. Among several kinds of cyclodextrins, B-cyclodextrin provided the best results. It was found that the optimum form of cyclodextrin utilization was the external addition of iclusion complexes between digitoxin and B-cyclodextrin at 1: 2 molar ratio from the beginning of biotransformation. With the optimized conditions, addition of B-cyclodextrin enhanced the production of digoxin up to 1.55 fold. In this case, not only digitoxin consumption was increased, but also the production of by-product was reduced.

The antitumor effects of mice fed with cell lysate of Lactobacillus plantarum were studied. The abdominal cancer induced by Sarcoma-180 was markedly inhibited and the expected life span was extended by 60% for the Balb/c mice fed with L. plantarum cell lysate for two weeks. A similar result was obtained for the rat inoculated with Spontaneous Osteosarcoma(SOS). The primary tumor volume of SOS was reduced by 70% for the rats fed with L. plantarum cell lysate (100mg/kg/day) for one week before the inoculation of SOS, while only 42% for the rats fed with the same amount of cell lysate for one week after the inoculation of tumor cell line, SOS. As lung was the metastasis site of SOS, the weight of lung was measured to determine the degree of metastasis inhibition by the L. plantarum cell lysate feeding. The rats fed with cell lysate for one week showed a remarkable inhibition of lung metastasis by 63%(before) and 46%(after), respectively. These results indicate that the feeding of L. plantarum cell lysate to mouse or rat can induce a strong stimulation of mucosal or systemic immune system and these effects results in an efficient antitumor activity.

The optimum conditions of the production of exo-polymer by Ganoderma lucidum WK-003 and it's hepatoprotective effect was studied. Optimum conditions for the production of exo-polymer (3.18 g/l) by using shaken flask culture of G. lucidum WK-003 were pH 4.5, 30℃, 120 rpm for 18 days cultivation. Also exo-polymer productio (7.15 g/l) was optimized by 5l jar fermenter cultivation with condition of pH 4.5, 30℃, 200 rpm, 1.0 vvm for 6 days cultivation. Glutamic pyruvic transaminase(GPT) activities in serum of intoxicated Sprague-Dawley rat were decreased from 704 IU/L to 330 IU/L by oral administration of the exo-polymer (20mg/kgday)f or 4 consecutive days.

To analyze the unique aspects of biomolecular processing for monoclonal antibody (MAb) production and secretion, the simple working model based on 3-compartment (endoplasmic reticulum, Golgi apparatus, and extracellular medium) was developed. Based on in vitro MAb assembly experimental results, the kinetic model for MAb assembly in the endoplasmic reticulumn was proposed. The dynamics of MAb assembly and secretion was simulated using methematica program. According to the simulation results, the proposed 3-compartment model provides an efficient means to predict the specific MAb productivity as well as intracompartmental concentrations of MAb in endoplasmic reticulum, Golgi apparatus, and extracellular compartment model. In vivo profiles of MAb intermediates gave good agreements with the simulation profiles predicted by the intracellular compartment model. Furthermore, results of such analysis can help in directing the control strategy for optimum biomolecular processing in a mammalian cell culture system.

Agar hydorlysates or agarooligosaccharides from agar prepared by Cytophaga agarase showed eight spots on TLC plate and the degree of polymerization of the spots were in the reange of 2.5 - 6.5. Each component of the hydrolysate as tested the several functionalities such as antimicrobial activity, anticavity activity, and anticoagulant activity. The anticativity activity and anticoagulant acitvity were found in all fractions of hydrolysates and several spot on TLC, whereas the anticoagulant activity was very low

Bacillus subtilis DT134 was resistant to 50-fold higher concentration of cadmium ions (Cd2+) than cadmium-sensitive B. subtilis BD224 in Luria Broth (LB) medium. Minimal inhibition concentration test in LB agar plates also showed similar results. The elevated cadmium resistance of B. subtilis DT134 strongly suggested a possible existence of cadmium resistance gene in it. Southern blot with Staphylococcus aureus cadA gene fragment (757 bp NlaIV-XmnI cadA DNA fragment) as probe was carried out to test the existence and similarity of the gene. In high stringency condition, there was no detectable signal, but in low stringency, a strong signal specific to the cadA probe could be detected. These results strongly suggested that there was some similarity between total DNA of B. subtilis DT134 and S. aureus pl258 in terms of cadmium resistance gene and the resistance mechanism might be an efflux mechanism. The subsequent efflux experiment showed that the cadmium resistance mechanism of B. subtilis DT134 was also due to the efflux of cadmium.

The effect of culture medium copper availability on the specific growth rate() and carbon conversion efficiency (CCE) was sutided for an obligatory methanotroph Methylosinus trichosporium OB3b under various combinations of carbon and nitrogen sources. Methane or methanol was used as a carbon source, and nitrate or ammonium was used as a nitrogen source. Medium copper availability determined the intracellular location or kind of methane monooxygenase (MMO), cell-membrane (particulate or pMMO) when copper was present and cytoplasm (soluble or sMMO) when copper was deficient. When methane was used as a carbon source, copper-containing medium exhibited higher and CCE than copper-free medium regardless of the kind of nitrogen source. When methanol was used as a carbon source, however, the effect of copper disappeared. Ammonium gave the higher and CCE than nitrate for both methane and methanol. Those observation suggest that there exist an important difference in energy utilization efficiency for methane assimilation between sMMO and pMMO.

This study was carried out to optimize the concentration of Ca2+ and pH of fusion medium which affected electrofusion frequency of protoplasts isolated from Nicotiana tabacum L. (cv. BY4) mesophyll cells and callus. The protoplasts were electrofused in the fusion media containing two different Ca2+ concentrations and three different pH regions. Fusion frequency was lower in the fusion medium containing only 13% mannitol as osmotic stabilizer. However, higher degree of fusion frequency (47.3%) was observed in the fusion medium containing 50mM CaCl2 at pH 10.5 than any other conditions. Cell viability was decreased by Ca2+ and high pH treatment in the fusion media, while fusion frequency was increased. It is concluded that Ca2+ is involved in electrofusion of protoplasts.

CHO 세포외 성장 및 생산성을 저해하는 엄포늄 이온의 동시 제거를 위해 PhJlhpsite~Gismondine 합성 zeolite가 alginate, cellulose acetate. dialysis membrane에 고정화된 흡착제가 개발되었다. 고정화 흡착체에 의한 암모늄 이온의 제거 효율 및 비에 따른 세표성장의 증진효과를 비교한 결과 최적의 고정화 흡착제로 membrane type이 선정되었다. 암모늄 이온의 제거 효파를 더욱더 명확하게 하고 고멸도의 세포 배양을 모사하가 위하여 8mM의 ammonium chloride를 첨가하고 membrane type 고정화 흡착제를 투여하여 암모늄 이온의 동시제거 효과를 조사한 결과 최대 세포 농도가 3배 이상 증가하였으며 생존율 역시 증가하였고 40%정도의 tPA 생산성 향상올 보여 주었다­. 이러한 증진 효과는 암모늄 이온의 고농도 시스템일수록 커졌다. 고정화 흡착제의 최적 투며 시기를 조사해본 결과 ammonium chlonde를 첨가하지 않았을 때 membrane type 고 정화 흡착제의 최적 투여 시기는 배양 시작 후 48시간이었고, 8mM의 ammonium chloride를 침가한 경우의 최적 투여 시기는 배양 시작 후 72시간이었다.

Three types of adsorbents were developed by immobilizing synthetic zeolite, Philipsite-Gismonine, in alginate, cellulose acetate and dialysis membrane for the in situ removal of ammonium ion which inhibits growth and productivity of animal cells such as CHO cells producing tPA. Ammonium ion removal efficiency and cell growth promoting effect with various immobilized adsorbents were evaluated and the membrane type was selected as an optimal immobilized adsorbent. The experiments were then simulated by adding 8mM ammonium chloride and immobilized adsorbent in order to validate the removal effect under high density cell cultures. The results showed increase in maximum cell density by three times, in cell viability, and in tPA productivity by 40%. And it was found that the promoting effects were more significant in case of high ammonium ion concentration system. It was also found that the optimum addition time for immobilized adsorbents was 48 hr in the absence of ammonium chloride addition and 72 hr in the presence of ammonium chloride addition.

The possibility of application of membrane type immobilized adsorbent to the fed-batch or perfusion culture system with anchorage-independent cells as well as batch system was investigated. The improvement in cell density and cell viability due to the combination of immobilized adsorbent with each culture system was evaluated for the investigation, and the optimum culture system employing immobilized adsorbent system was suggested based on the results. It was observed that the system with immobilized adsorbent showed better cell growth and cell viability than that without immobilized adsorbent in every operation system of batch, fed-batch, and perfusion. In case of batch system, 200% improvement of maximum cell density was observed in the system where ammonium chloride was added on purpose. And 50% improvement of maximum cell density was observed in the fed-batch system where ammonium ion accumulates significantly, while small increase in maximum cell density was observed in the perfusion system where dilution of waste byproducts exists. Especially, the fed-batch system showed the most significant improvement on cell growth because both compensation of nutrient and removal of ammonium ion occurred simultaneously in the system. Therefore a combined system of immobilized adsorbent and fed-batch operation could be suggested as an optimum system with in situ removal of ammonium ion.

Cellular transfections with cationic liposomes are widely empolyed for gene and oligonucleotide transfer in vitro because of their safety and ease of use. However, they still suffer from the low transfection efficiency comparing with viral vectors. Substantial effort shave been focused on increasing transfection efficiency by supplementing the liposome/DNA complexes(lipoplex) with various components. In this work, we tired three kinds of supplements, Poly-L-lysine(PLL), transferrin and a mixture of anionic lipids(PS/PE/PC), to study their effects on gene transfer yield and gene expression efficiency. PLL, a polycationic polymer, enhanced gene transfer yield by 3 times but the gene expression efficiency was increased only by 1.5 times. this result implies that PLL can enhance the transfection efficiency mainly by increasing the rate of outermembrane transport of lipoplex into the cells. On the other hand, transferrin which can facilitate the gene transfer via ligand-receptor interaction gave not only increased gene transfer yield but also enhanced gen expression efficiency by 2.8 times. Transferrin seems to contribute to the escape of plasmid from endosomes through ligand-receptor recycle mechanism. When the cells were treated with a mixture of anionic lipids for 3 hours before the transfection, gene transfer yield was slightly decreased but the gene expression efficiency was enhanced by 1.9 times. This is presumably due to the accelerated liposome-plasmid dissociation by the anionic lipids, and the increased delivery of plasmid to the nucleus. According to these results, it is clear that the supplementation to ameliorate transfection efficiency with cationic liposomes should be contrived in the direction of increasing delivery of plasmid.

Immunositmulation effects of the mice fed with the cell lysate of Lactobacillus plantarum isolated from Kimchi were studied. The mice group fed with cell lysate was different from the control group on the degree of immune responses, e.g. 1) proliferation of splenocytes and Peyer's patch cells, 2) production of nitric oxide (NO) by peritoneal macrophages, 3) production of intestinal secretory lgA (slgA), 4) variation of TNF- and IL-2 concentration in blood, 5) production of specific lgG against sheep red blood cells. A general enhancement in enteric and systemic immune responses was observed with a simple oral administration of immunostimulators. With the oral feeding of L. plantarum, not only the total amount of gut secretion antibody, but also the binding capacity of antibodies to the enteric microorganisms including L. plantarum was increased. These experimental results clearly showed that the oral feeding of immunostimulators gave multifunctional effects on the mucosal and systemic immune systems of mice.

Optimal media and cultural conditions for the production of prodigiosin-like pigment were established using Serratia sp. KH-95. Glucose and phosphate(K2PO4) stimulated the cell growth, but inhibited the production of pigment at concentration levels of above 10 g/L and 2.0 g/L, respectively. Addition of soy been oil or rice oil to the production medium accelerated cell growth up to more than 2-3 times, but the production of prodigiosin increased about 15-20% in spite of the good cell growth. The effect of pH on the production of pigment was investigated in a 5 liter-bioreactor. When the pH of culture broth was maintained below 8.0, most of pigment was attached to the surface of cells. When the pH of culture broth was above 8.5, however, about 70% of total pigment was suspended in the supernatant of the broth. The cell growth and production of pigment were inhibited at dissolved oxygen concentration of below 10% of air-saturation.

Effects of various environmental factors on cell size index(FCW/DCW) in Thalictrum rugosum. Lithospermum erythrorhizon and Taxus cuspidata plant cell suspension cultures were investigated. Time course change of cell size index were also observed. In batch cultures, FCW/DCW increased according to the decrease of sugar concentration. For short-term experiment within 24 hr, FCW/DCW value could be reduced significantly by increasing sugar concentration. When an osmoticum such as mannitol was added, FCW/DCW converged to a low value. Therefore, it was confirmed that osmolality of the medium was important in determining cell size or water content of the cells. Inorganic salts or treatment with organic solvent also exhibited some effect on the cell size index. However, pH and centrifugal force did not show any influences. On the other hand, it was found that the addition of Pluronic F-68 reduced FCW/DCW. By combining these results effectively, it may be possible to increase the cell concentration in high density culture to a higher extent.

Biosorption of Pb and Cu was evaluated for 23 species of marine algae collected from a Korean coast. Among a variety of species for biosorbent potential, Hypnea charoides showed the highest capacity for Pb. An adsorption equilibrium was reached in about 2 hr for Pb and 30 min for Cu. The uptake capacity was 192.8 mg Pb/g biomass and 256 mg Cu/g biomass, respectively. The adsorption parameters for Pb and Cu were determined according to Langmuir model. With an increase in pH value, more negative sites are becoming avaliable for adsorption of pH and Cu, thus the removal of Pb and Cu increases at alkaline conditions. The selectivity of mixture solution shows the uptake order of Pb>Cu>Cr>Cd. When Ca concentration increases in Pb solution, Pb was selectively adsorbed.

The bacterial motility in sand was studied with Alcaligenes xylosoxidans Y234 which is known as a strong decomposer of aromatic chemicals, especially toluene. Apparent motility coefficient (c,app) and apparent chemotaxis coefficient (uc,app) for toluene were measured in the sands which have four different porosities. Adsorption ratio of Alcaligenes xylosoxidans Y234 on the sands was measured as 17%. The ramdom motility coefficients were 0.85∼1.68x10-3cm2/sec, and decreased as the porosity of sands decreased. Apparent chemotaxis coefficients were measured as 1.1∼6.8x10-5cm2/sec, and decreased as the porosity decreased and with time. The tendency of alcaligenes xylosoxidans Y234 movement towards toluene seemed very weak and showed little chemotaxis.

Fed-batch fermentations were carried out in order to improve the efficiency of hirudin production by recombinant Saccharomyces cerevisiae. A fed-batch fermentation done with the optimized semi-synthetic medium resulted in a maximum hirudin concentration of 342mg/l by keeping a galactose concentrations between 10 and 30g/l which corresponded to a 11.4-fold increase in hirudin concentration compared with the simple bach fermentation done with the same medium. Comparison of the chromatographic pattern of proteins in the growth medium clearly showed that the use of the semi-synthetic medium is more advantageous for separation of hirudin than the case o fusing the complex medium. Continuous cell recycle fermentation done at dilution rate of 0.1h-1 and an inlet galactose concentration of 100g/l yielded a maximum hirudin productivity of 19.1mg hirudin/lh

A fermentation system was supplemented with a device for the measurement of the durations of alkali pump feeding for automatic pH control and an A/D convertor for precise monitoring of pH value by computer. A software program was developed to measure buffer capacities from the pH signal and the pH control signal during fermentation. By measuring the buffer capacity on-line, levels of acetic acid were estimated by a software sensor using pH signal in a fermentation process of E.coli growing in a minimal medium. The measured values of acetic acid showed correlation to those of estimated by the software sensor. Lacitic acid production was also successfully estimated by the values of buffer capacities measured on-line.