• The Korean Journal of Pharmacology
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• v.32 no.3
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• pp.433-444
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• 1996

We describe a novel approach to evaluate quantitatively the amounts of denatured proteins in cells upon heat exposure. A thiol compound, diamide [azodicarboxylic acid bis (dimethylamide)] causes protein cross-linking with exposed sulfyhydryl residues of denatured proteins. Since denatured proteins expose normally well-hidden sulfhydryl groups, these will be preferentially cross-linked by diamide. Thus diamide acts to 'trap' denatured proteins. We observed that protein aggregates (high molecular weight protein aggregates, HMA) appeared on SDS-polyacrylamide gels run under non-reducing conditions and that the amount of HMA can be quantified by scanning the gels using a gas flow counter. Heating cells followed by a fixed dose of diamide exposure resulted in HMA increases in a heat-dose dependent manner, demonstrating that the quantitation of HMA could serve as a measure of heat-denatured proteins. We compared thermotolerant and nontolerant cells and found decreased HMA in tolerant cells upon heat treatment. As an attempt to examine the kinetics of protein renaturation (or 'repair'), we measured the amounts of aggregates formed by the addition of diamide at various times after heat shock. Such experiments demonstrate an equally rapid disappearance of HMA in previously unheated and in thermotolerant cells. Levels of HMA in tolerant cells increased significantly after electroporation of HSP70 specific mAbs, suggesting an involvement of HSP70 in reducing HMA levels in thermotolerant cells upon heat exposure. Immunoprecipitation studies using anti-HSP70 antibody indicated an association of HSP70 with heat-denatured proteins. Our results suggest that heat induces protein denaturation, and that elevated level of HSP70 present in thermotolerant cells protects them by reducing the level of protein denaturation rather than by facilitating the 'repair' (or degradation) process.

• Microbiology and Biotechnology Letters
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• v.40 no.4
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• pp.339-347
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• 2012

Viral safety is an important prerequisite for clinical preparations of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacturing process. In particular, Chinese hamster ovary (CHO) cells are highly susceptible to several RNA viruses including reovirus (Reo), bovine viral diarrhea virus (BVDV), and bovine parainfluenza virus (BPIV) and there have been reports of such viral contaminations. Therefore, viral detection during the CHO cell process is necessary to ensure the safety of biopharmaceuticals against viruses. In this study, a multiplex reverse transcription (RT)-PCR assay was developed and subsequently evaluated for its effectiveness as a means to simultaneously detect Reo, BVDV, and BPIV during the manufacture of cell culture-derived biopharmaceuticals. Specific primers for Reo, BVDV, and BPIV were selected, and a multiplex RT-PCR was optimized. The sensitivity of the assay for simultaneous amplification of all viral target RNAs was $7.76{\times}10^2\;TCID_{50}/ml$ for Reo, $7.44{\times}10^1\;TCID_{50}/ml$ for BVDV, and $6.75{\times}10^1\;TCID_{50}/ml$ for BPIV. The multiplex RT-PCR was proven to be very specific to Reo, BVDV, and BPIV and was subsequently applied to the validation of CHO cells artificially infected with each virus. It could detect each viral RNA from CHO cells as well as culture supernatants. Therefore, it was concluded that the multiplex RT-PCR assay can be applied to detection of the adventitious viruses during the manufacture of cell culture-derived biopharmaceuticals.

• The korean journal of orthodontics
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• v.34 no.6 s.107
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• pp.506-513
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• 2004

Osteoclast action is necessary for alveolar bone remodeling in orthodontic tooth movement. The nervous system has also been reported to be associated with bone remodeling. This study was aimed to investigate the changes of osteoclasts in the periodontal ligament (PDL) space after surgical resection of the inferior alveolar nerve (IAN). Experimental rats were divided into young and adult groups. A surgical resection procedure of the IAN was carried out in the left side of the mandible and a sham operation in the right side of the mandible. The number of osteoclasts on the bundle bone surface and the resorption activity of the osteoclasts were histomorphometrically measured. The changes in distribution of substance P (SP) immunoreactive (IR) nerve fiber were evaluated in the PDL and pulp. SP-IR nerve fiber was depleted in both the PDL and pulp of the IAN resection side in both groups, which confirmed the resection of IAN to be successfully conducted. The number of osteoclasts in the IAN resection side was significantly reduced in both the young and adult groups (p<0.01 and p<0.05), whereas the resorption activity of osteoclasts did not show any significant difference between the IAN resection side and the sham operation side in both groups (p>0.05 and p<0.05). The adult group showed that the number of osteoclasts reduced significantly (p<0.01) and the resorption activity didn't change in comparison with the young group (p>0.05). These results suggest that surgical resection of the IAN and aging reduce the population of the recruited osteoclasts within the PDL, but don't affect on the osteoclastic resorption activity.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.4
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• pp.420-426
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• 1999

We examined the immune response in flounder, Paralichthys olivaceus, with immunization of formalin killed Edwardsiella tarda as an antigen. The ELISPOT-assay (enzyme-linked immunospot assay) was optimized technically and applied to count the number of total and specific antibody secreting cells (TASC and SASC) in lymphocytes of different lymphatic organs. Incubation of lymphocytes on 96 well plate for more than 2.5hrs came out enough time in ELISPOT-assay for counting the antibody secreting cells in the anterior kidney and spleen. However, too much of plate-coated antigen or rabbit anti-flounder immunoglobulin for SASC or TASC counting, respectively, was appeared to decrease the sensitivity of the assay system. Specificity of the system was also confirmed by the absence of TASC in lymphocytes treated with cycloheximide to prevent protein synthesis. The peak numbers of SASC appeared at wk 3 post immunization after that there was a sharp decrease and reached to almost zero at wk 7. In the spleen and kidney, the timing and numbers of SASC on peak response were concurrent without preferential organ distribution. The specific antibody level in the sera increased rapidly between wk 2 and 3 after immunization, i.e. like the specific cellular response found with ELISPOT-assay on that period, However, the remained high level of specific serum antibody from wk 5 after immunization until the end of experiment was clearly distinguishable from the kinetics of SASC response decreased sharply.

• Journal of the Korean Society of Food Science and Nutrition
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• v.36 no.9
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• pp.1099-1105
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• 2007

The present study describes the preliminary evaluation of the anticancer activity of Styela plicata. Freeze-dried S. plicata was extracted with methanol, ethanol, acetone, and water, and then anticancer effect of the extracts was measured by the MTT reduction assay and phase-contrast microscopy on the HT-29 human colon carcinoma cells. Among the extracts, acetone extract showed the highest anticancer activity. The cell proliferation rates markedly decreased by 94.0% at the concentration of 500 ${\mu}g/mL$ of acetone extract compared with control cells. The acetone extract was further fractionated with hexane, diethyl ether, ethyl acetate, and water layer according to the degree of polarity. The HT-29 cells with hexane layer extract (250 ${\mu}g/mL$) decreased the cell viability to 5.1% of untreated control. The growth of SW620, HeLa, and MCF-7 cells was decreased to about 10%, by the treatment of hexane layer extract 250 ${\mu}g/mL$. Theses results suggest extracts from S. plicata as possible natural cancer therapeutic material.

• Korean Journal of Medicinal Crop Science
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• v.7 no.2
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• pp.129-137
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• 1999

The structure of amyloplasts and intercellular transport in the old and new bulbs of Frjtillaria pallidiflora were observed by means of electron microscope. The structure of internal membrane system was different between new and old amyloplasts. The active intercellular transport was observed in both new and old bulbs. The phenomena of encytosis and exocytosis always could be found in the cell membrane, and plasmodesmata established a symplasmic pathway for intercellular transport. Groups of vesicles often located at the ends of plasmodesmata, showing that they participated in the intercellular transport. These results laid a foundation for the further study on the mechanism of growth and development in Fritillaria pallidiflora.

• Applied Microscopy
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• v.35 no.2
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• pp.73-80
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• 2005

To observe the morphological changes and the expression of tenascin in synovium of human knee joint, between normal condition and degenerative arthritis, were processed by immunoelectron microscopic method. The results were summarized as follows. 1. In degenerative arthritis, the hyperplasia of synovial membrane was characterized by the increase of cell number in secretory synovial cells. 2. In normal condition, there was no marking of the immuno-gold for tenascin in synovial membrane. 3. In degenerative arthritis, the immuno-gold for tenascin were observed in endoplasmic reticulum of secretory synovial cells and extracellular matrix of synovial layer. On the basis of above findings, the hyperplasia of synovial membrane and the pathologic processes may be concerned with the increase of number of secretory synovial cells and of expression of tenascin, in degenerative arthritis.

• Proceedings of the Korean Society of Postharvest Science and Technology of Agricultural Products Conference
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• 1994.07a
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• pp.7-8
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• 1994

한국산 표고버섯 균사체를 액체배양하여 천연항암물질로 알려진 단백다당체를 추출한 후 그 물질의 특성에 대하여 검토하였다. 균사체의 최적 재양조건을 TGY배지로 조사한 바 온도 $25^{\circ}C$, 배양초기 pH4.0, 교반속도 300rpm, 균사배지 접종량을 10.0%로 하고 산소 통기량을 1.0volume of ait/volume of medium/mimute으로 하였을때 가장 양호한 조건이였으며, 대량생산 하기 위한 SCM배지에서 최적의 C/N비는 13.1오써 7일간 배양하였을때 18.8g/L의 균사령을 얻었으며, 이때 생산수율은 0.46으로 나타났다. 발효가 끝난 배양액에서 균사, 여액 그리고 배양액의 전체에서 단백다당체를 분리한 결과 각 분획에서 단백다당체가 각각 0.55%, 0.12%, 0.69%가 회수되어 배양액 전체에서 단백다당체를 추출하는 것이 바람직하며, 추출방법으로 열수추출, glass bead추출 및 cellulasa처리를 하여 단백다당체의 수율을 비교한 결과 0.25-0.5mm glass nead로 30분간 균사체를 분쇄한 다음 열수추출을 1시간을 하였을때 990mg/100ml의 단백다당체를 얻을 수 있었다. 고단백다당체를 1차 단백질 가수분해 효소로 분해하고, EDAE cellulose 및 Sepadex G-100 column chromatography로 정제한 후, TLC/FLD, ultracentrifugation한 결과 순수한 물질임을 알 수 있었다. 단백다당체의 항암효과 조사중 in vitro배양에서 $P_{388}$과 $L_{1210}$에 대한 단백다당체의 활성단위 1 unit는 1mg정도였으며, 인체의 장암세포인 HCT-48, HRT-18, HT-29 밀 간암세포인 Hep G2 대한 생육저해 단위는 각각 4.4, 3.6, 6.6, 2.6mg이었다. HCT-48과 Hep G2 세포의 크기 분포도는 대조군에 비하여 시간이 경과함에 따라, 그리고 단백다당체의 농도가 증가함에 따라 peak가 작은 size 쪽으로 이동하였다. 또, 단백다당체를 첨가 배양한 HCT-48과 Hep G2세포의 현미경 관찰에서 본래의 암세포 형태가 변형되고 크기가 감소하며 세포사이의 경계막이 흐트러지면서 세포수가 감소하고 사멸하였다. In vivo실험에서는 대조군보다 단백다당체를 첨가한 군에서 항체 형성능력이 대조군에 비하여 형질세포가 2배로 증가하였다. 단백다당체의 화학적 성분 분석에서 다당함량은 46.1%이면 구성다당류는 glucose, galactose, mannose, xylose로 구성되었고 단백질의 함량은 7.28%이며, 구성아미노산은 15종의 아미노산으로 되었다. 또 무기물은 Na, K, Zn, Ca등의 순으로 이루어 짐을 알 수 있었다.

• The Journal of Korean Obstetrics and Gynecology
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• v.18 no.1
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• pp.45-54
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• 2005

이 연구는 SKBR3 인간 유방암세포주에 대한 귀전우 메탄올추출물(CME)의 증식억제 효과, 세포사 유발 효과 및 항산화 활성을 확인하기 위해 이루어졌다. SKBR3 유방암세포주는 48시간동안 다양한 농도($0{\sim}20{\mu}g/m{\ell}$)의 CME가 제공된 곳에서 배양되었고, MMT 측정법을 이용하여 세포생존율을 평가하였다. CME의 50%에서 효과를 나타내게 하는 약물농도인 $ED_{50}$ (effective dose 50%)은 $6.5{\pm}0.3{\mu}g/m{\ell}$) 이며, 투여량이 증가함에 따라 농도에 의존하여 세포증식이 억제되는 것으로 나타났다. 또한 CME의 증식억제 효과는 유방암세포주의 세포사와 관련됨의 세포의 형태학적 변화와 올리고뉴클레오솜 DNA 파편의 확인을 통해 알 수 있었다. 또한 다양한 농도와 배양시간에서 CME가 ROS의 생산을 억제한다는 것을 확인할 수 있었다. 이런 결과들은 귀전우의 메탄올추출물이 SKBR3 인간 유방암세포주에 대해 강력한 증식억제 효과와 강한 항산화 효과를 나타낼 뿐만 아니라 세포사를 유도하는 효능을 가지고 있음을 시사한다. 이러한 효능은 약물에 대한 노출시간과 투여량에 의존하였다. 따라서 귀전우는 다양한 기전에 의해 유방암 세포에 대한 억제효과를 가질 수 있을 것으로 인식할 수 있다.

• Parasites, Hosts and Diseases
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• v.29 no.4
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• pp.371-380
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• 1991

The effects of peritoneal exudate cells(PEC) and sera of athymic nude and DS mice infected with Clonorchis sinensis metacercariae or sensitized by injection of metabolic products into footpad on transfer of immunity against the fluke to the syngeneic mice were studied. There was no significant difference in eggs per gram pattern between the sensitized and control groups, and between nude and DS mice. However, the worm burdens were slightly greater in nude mice than in DS mice. Also, a few plaque forming cells were found in only DS mice given PEC and serum from Group II DS mice. In the light of these results, it is likely that PEC and sera of nude or DS mice which are deficient, at least partially, in the cellular immune system are unable to transfer immunity against C. sinensis to syngeneic recipients.