• The Korean Journal of Physiology and Pharmacology
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• 제24권3호
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• pp.267-276
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• 2020

T In the present study, we investigated the effect of oncogenic H-Ras on rat mdr1b expression in NIH3T3 cells. The constitutive expression of H-Ras^V12 was found to downregulate the mdr1b promoter activity and mdr1b mRNA expression. The doxorubicin-induced mdr1b promoter activity of the H-Ras^V12 expressing NIH3T3 cells was markedly lower than that of control NIH3T3 cells. Additionally, there is a positive correlation between the level of H-Ras^V12 expression and a sensitivity to doxorubicin toxicity. To examine the detailed mechanism of H-Ras^V12-mediated down-regulation of mdr1b expression, antioxidant N-acetylcysteine (NAC) and NADPH oxidase inhibitor diphenylene iodonium (DPI) were used. Pretreating cells with either NAC or DPI significantly enhanced the oncogenic H-Ras-mediated down-regulation of mdr1b expression and markedly prevented doxorubicin-induced cell death. Moreover, NAC and DPI treatment led to a decrease in ERK activity, and the ERK inhibitors PD98059 or U0126 enhanced the mdr1b-Luc activity of H-Ras^V12-NIH3T3 and reduced doxorubicin-induced apoptosis. These data suggest that Ras^V12 expression could downregulate mdr1b expression through intracellular reactive oxygen species (ROS) production, and ERK activation induced by ROS, is at least in part, contributed to the downregulation of mdr1b expression.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1995년도 제3회 추계심포지움
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• pp.107-113
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• 1995

Basement membrane laminin is a multidomain glycoprotein that interacts with itself, heparin and cells. The distal long arm plays major cell and heparin interactive roles. The long arm consists of three subunits (A, B1, B2) joined in a coiled-coil rod attached to a terminal A chain globule (G). The globule is in turn subdivided into five subdomains (Gl-5). In order to analyze the functions of this region, recombinant G domains (rG, rAiG, rG5, rGΔ2980-3028) were expressed in Sf9 insect cells using a baculovirus expression vector. A hybrid molecule (B-rAiG), consisting of recombinant A chain(rAiG) and the authentic B chains (E8-B)was assembled in vitro. The intercalation of rAiG into E8-B chains suppressed a heparin binding activity identified in subdomain Gl-2. By the peptide napping and ligand blotting, the relative affinity of each subeomain to heparin was assigned as Gl> G2= G4> G5> G3, such that G1 bound strongly and G3 not at all. The active heparin binding site of G domain in intact laminin appears to be located in G4 and proximal G5. Cell binding was examined using fibrosarcoma Cells. Cells adhered to E8, B-rAiG, rAiG and rG, did not bind on denatured substrates, poorly bound to the mixture of E8-B and rG. Anti-${\alpha}$6 and anti-${\beta}$1 integrin subunit separately blocked cell adhesion on E8 and B-rAiG, but not on rAiG. Heparin inhibited cell adhesion on rAiG, partially on B-rAiG, and not on E8. In conclusion, 1) There are active and cryptic cell and heparin binding activities in G domain. 2) Triple-helix assembly inactivates cell and heparin binding activities and restores u6131 dependent cell binding activities.

• 대한한의학회지
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• 제20권2호
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• pp.165-176
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• 1999

The in vivo effects of Fructus Benincasae (FB-Baekdongkwa(B) and FB- on the expectoration (decrease in sputum viscoelasticity) by their sorts and using rats (Sp. D. male, $150{\sim}160g$). FB was divided by seed, flesh, and bark, extracted by 95% ethanol for 3 hr. The extracts were given to rats administration and the following results were obtained: 1. When FB Recens-C extract was administered at the concentration of 300 mg/kg b.w., mucus secretion effect in the trachea was desirably stimulated. 2. The secretion of phenol red was increased in the FB-treated tracheas in the order of Semen Benincasae(SB)-B $(153{\pm}8\;%)$, FB-B $(149{\pm}10\;%)$, and FBR-C $(117{\pm}26\;%)$. In general, the effect of FB-B extract on phenol red secretion was stronger than that of FB-C. 3. When tracheobronchial lavage fluid was analyzed, the mucus secretion was relatively high $(111{\pm}14\;%)$ in FB-B compared with other extracts. 4. Microscopic analysis after direct treatment of the FB extracts to the rat tracheal tissue showed that all the FB extracts possessed no effects for the activity of the ciliary movement. 5. Glycoprotein content secreted by the seed extract of FB-B was increased compared with the control group, which represents the highest secretion effect of mucus. From the above results. we could conclude that the seed of SB-B possesses better activity for mucus secretion from trachea than the extracts of any other parts. Therefore, it is expected that the seed of SB-B may be available for the purpose of expectorant activity in the prescription of traditional medicine.

• Asian-Australasian Journal of Animal Sciences
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• 제22권1호
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• pp.20-25
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• 2009

Xenotransplantation of pig organs into humans is a potential solution for the shortage of donor organs for transplantation. However, multiple immune barriers preclude its clinical application. In particular, the initial type of rejection in xenotransplantation is an acute cellular rejection by host $CD8^+$ cytotoxic T lymphocyte (CTL) cells that react to donor major histocompatibility complex (MHC) class I. The human cytomegalovirus (HCMV) glycoprotein Unique Short (US) 2 specifically targets MHC class I heavy chains to relocate them from the endoplasmic reticulum (ER) membrane to the cytosol, where they are degraded by the proteasome. In this study we transfected the US2 gene into minipig fetal fibroblasts and established four US2 clonal cell lines. The integration of US2 into transgenic fetal cells was confirmed using PCR and Southern blot assay. The reduction of Swine Leukocyte Antigen (SLA)-I by US2 was also detected using Flow cytometry assay (FACS). The FACS analysis of the US2 clonal cell lines demonstrated a substantial reduction in SLA-I surface expression. The level (44% to 76%) of SLA-I expression in US2 clonal cell lines was decreased relative to the control. In cytotoxicity assay the rate of $CD8^+$ T cell-mediated cytotoxicity was significantly reduced to 23.8${\pm}$15.1% compared to the control (59.8${\pm}$8.4%, p<0.05). In conclusion, US2 can directly protect against $CD8^+$-mediated cell lysis. These results indicate that the expression of US2 in pig cells may provide a new approach to overcome the CTL-mediated immune rejection in xenotransplantation.

• Journal of Microbiology and Biotechnology
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• 제12권3호
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• pp.463-469
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• 2002

Rotaviruses are the world-wide leading causative agents of severe dehydrating gastroenteritis in young children and animals. The outer capsid glycoprotein VP7 and inner capsid glycoprotein VP6 of rotaviruses are highly antigenic and immunogenic. An SFV-based expression system has recently emerged as a useful tool for heterologous protein production in mammalian cells, exhibiting a much more efficient performance compared to other gene expression systems. Accordingly, the current study adopted an SFV-based expression system to express the VP7 of a group A human rotavirus from a Korean isolate, and the VP6 of a group B bovine rotavirus from a Korean isolate, in mammalian cells. The genes of the VP6 and VP7 were inserted into the SFV expression vector pSFV-1. The RNA was transcribed in vitro from pSFV-VP6 and pSFV-VP7 using SP6 polymerase. Each RNA was then electroporated into BHK-21 cells along with pSFV-helper RNA containing the structural protein gene without the packaging signal. The expression of VP6 and VP7 in the cytoplasm was then detected by immunocytochemistry. The recombinant virus was harvested by ultracentrifugation and examined under electron microscopy. After infecting BHK-21 cells with the defective viruses, the expressed proteins were separated by SDS-PAGE and analyzed by a Western blot. The results indicate that an SFV-based expression system fur the VP6 and VP7 of rotaviruses is an efficient tool for developing a diagnostic kit and/or preventive vaccine.

• 한국축산식품학회지
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• 제21권4호
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• pp.367-373
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• 2001

MFGM 당단백질의 하나인 PAS-7을 GPC 및 affinity chromatography에 의해 정제하여 2-AB로 형광 표식한 후, anion-exchange column 및 reversed-phase column을 이용해 5개의 성분을 분리하였다. 그 중 가장 상대량이 많은 성분 e에 대하여 RAAM2000을 이용한 당쇄구조 해석을 실시하여, 성분 e는 RAAM2000 GPC에 의하여 4개의 성분으로 분리되어 각각 calibration standard 12.10, 8.88, 5.84 및 4.86GU의 용출위치에 검출되었다. 이 용출위치와 당쇄구조는 livrary의 component-7457과 일치하며, 12.2GU의 크기로 분자량은 1788로 판단되며 library의 당쇄와 약 85%의 확률로 일치했다. 그 결과 성분 e의 당쇄구조는 환원말단에 $\alpha$1-6결합된 fucose를 1개 함유하며, core부분의 비환원말단에 N-acetyllactosamine branch를 2개 함유한 전형적인 biantennary 당쇄구조인 것으로 추축되어, 이전 HPLC, acetolysis, sequential exoglycosidase 소화, NMR분석에 의해 보고된 성분 7N1A의 구조와 일치함으로써, OGS RAAM2000을 이용한 PAS-7의 당쇄구조 해석의 유용성이 증명되었다.

• 한국동물위생학회지
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• 제44권2호
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• pp.73-83
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• 2021

Although many swine farms continuously vaccinated to sow to prevent Porcine epidemic diarrhea(PED), PED has occurred annually in swine herds in Jeonbuk province, Korea. In the present study, the small intestine and feces samples from 17 farms where severe watery diarrhea and death of newborn piglets occurred in 2019 were collected, amplified by RT-PCR and determined the complete nucleotide sequences of the spike (S) glycoprotein genes of nine Jeonbuk PEDV isolates. The spike (S) glycoprotein is an important determinant for molecular characterization and genetic relationship of PEDV. These nine complete S gene isolates were compared with other PEDV reference strains to identify the molecular diversity, phylogenetic relationships and antigenicity analysis. 9 field strains share 98.5~100% homologies with each other at the nucleotide sequence level and 97.3~100% homologies with each other at the amino acid level. The nine Jeonbuk PEDV isolates were classified into G2b group including a genetic specific signal, S-indels (insertion and deletion of S gene). In addition, comparisons the neutralizing epitopes of S gene between 9 field strains and domestic vaccine strains of Korea mutated 12-15 amino acids with SM-98-1 (G1a group) and mutated 0-3 amino acids with QIAP1401 (G2b group). Therefore, the development of G2b-based live vaccines will have to be expedited to ensure effective prevention of endemic PED in Korea. In addition, we will need to be prepared with periodic updates of preventive vaccines based on the PEDV variants for the re-emergence of a virulent strain.

• 한국식품과학회지
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• 제30권4호
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• pp.976-982
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• 1998

고사리 수추출물에서 분리된 단백다당획분(Pteridium aquilinum glycoprotein, PAG)이 마우스 면역계에 미치는 영향을 알아보기 위하여 BALB/C 마우스의 복강내에 14일간 반복 투여한 후 마우스의 체중 및 면역장기의 무게변화를 관찰하면서 마우스 항체생성능과 비장세포 증식능에 대하여 조사하였다. PAG를 용량별$(0{\sim}500{\;}{\mu}g/kg)$로 투여한 결과 마우스 체중변화와 면역장기에는 커다란 효과를 관찰할 수 없었으나, HEL에 대한 항체생성능에 있어서는 $500{\;}{\mu}g/kg$ 투여시 뚜렷한 증가를 보였다. 시험물질을 투여한 마우스로부터 비장세포를 분리하여 phytohemagglutinin (PHA) 혹은 lipopolysaccharide (LPS)에 대한 비장세포 증식능을 측정한 결과, PAG 투여군에 있어서는 PHA와 LPS유도 비장세포 증식능이 용량 의존적으로 증가되었음이 관찰되었다. 비장세포 증식능에 대한 in vitro 시험결과 LPS에 대한 반응증가는 단백다당획분(PAG)의 세포에 대한 직접적인 작용에 의하며, PHA에 대한 반응증가는 세포에 대한 직접적인 작용은 아님을 알 수 있었다. PAG의 항체생성능 증가에 기여하는 활성부위를 규명하기 위하여 periodate산화 및 pronase소화를 실시하여 얻어진 각각의 조단백, 조다당분들과 고사리 수추출물에서 정제된 정제다당획분이 항체생성능에 미치는 영향에 대하여 시험하였다. 시험결과 조다당투여군과 정제다당 투여군에서는 항체생성능 증가가 관찰되지 않았으나 periodate 산화물인 조단백 투여군에서 뚜렷한 항체생성능을 확인할 수 있었으며, 이와 같은 실험결과 PAG의 단백성분이 항체생성능 증가에 있어서 주요 활성 본체임을 알 수 있었다., 660 mmHg 및 560 mmHg 압력 중 560 mmHg의 감압 하에서 5회 반복 수세하는 것이었다. 분자구조 변형에 의한 것이라 사료된다./TEX>일 경에 점질물과 산패취가 발생하여 상품으로서의 가치를 상실하였고, $-20^{\circ}C$ 저장구는 저장 100일 까지 상품으로서 가치를 유지하였다.등의 네 가지 요인으로 나누어서 각각의 효과를 요인간으로 비교하면 한일 양국이 함께 가격의 효과가 가장 작다. 그러나 그 이외 요인별 효과의 상대적 중요성은 양국간에 다른데 한국은 소비지출의 효과가 출생연도나 연령 효과보다 크지만 일본은 경제적 요인인 소비지출보다 세대주의 출생연도나 연령 등의 비경제적 요인의 효과가 크다.가능한 전분으로, 완전한 결정성구조를 가진 전분이 아닌 것으로 사료된다.147.33 peak area%인 것과 유사(類似)하게 높았다.tarrow}6$보다 $4{\beta}{\rightarrow}8$의 결합방식이, 그리고 procyanidin 류에서는 gallate를 갖는 물질이 이를 함유하지 많은 물질보다 더욱 높은 ACE의 저해효과를 나타내었으며, 또한 hydroxyl 기가 많을수록 효소 저해효과도 증가하는 것으로 나타났다. 이와 같은 결과는 chocolate의 주원료인 cacao bean의 polyphenol 성분 또한 녹차 등에서 볼 수 있는 생리활성효과에 손색없는 것으로 판단되고, 이러한 기능성에 기초하여 chocolate, 음료 등의 식품이나 의약품의 기능성 소재로서의 산업적 응용 가능성이 높은 것으로 사료된다.대장균군이 역시 난각에서 가장 높은 빈도로 분리되었고, 난황(Yolk)에서는 극히 낮은 수준의 세균오염도를 보였다. 다양한 동물종유래 S. aureus 균주들의 유전학적 분석목적에 가장 신뢰도 높고 감별능력이

• Asian-Australasian Journal of Animal Sciences
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• 제16권4호
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• pp.498-503
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• 2003

Luteinizing hormone as other glycoprotein hormones is characterized by a heterodimeric structure composed a common $\alpha$-subunit noncovalently linked to a specific $\beta$-subunit. The correct conformation of the heterodimer is important for efficient secretion, hormonal-specific post-translational modifications, receptor binding and signal transduction. To determine whether $\alpha$- and $\beta$- subunits can be synthesized as a single polypeptide chain (tethered-bLH and -bFSH) and also display biological activities, the tetheredbLH and -bFSH molecules were constructed and transfected into chinese hamster ovary (CHO-K1) cells. LH and FSH activities were assayed by using the human embryonic kidney (HEK) 293 cells expressing rat LH and FSH receptor genes. The tethered-bLH and - bFSH proteins were efficiently secreted and showed a similar activity to the dimeric bovine LH and FSH $\alpha$/$\beta$ wild type and native purified from bovine pituitary. The tethered-molecules can be permit development of potent new analogues that stimulate ovarian development. Taken together, a single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds. These data indicate the potentiality of the single chain approach to further investigate structurefunction relationships of LH and FSH.

• Journal of Microbiology and Biotechnology
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• 제1권4호
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• pp.232-239
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• 1991

Caboxymethyl cellulase (CMCase) I was purified from the induced culture filtrate of Penicllium verruculosum F-3 by ammonium sulfate precipitation, DEAE-Sephadex A-50 chromatography and Bio-gel P-150 filtration. The purified enzyme was assumed to be a glycoprotein consisting of 8.5% carbohydrate and having a molecular weight of 70.000 in SDS-polycrylamide gel electrophoresis (SDS-PAGE). The purified enzyme-specific anti-CMCase I IgG was obtained by rabbit immunization and protein A-sepharose CL-4B chromatography. The fungal poly($A^+$) RNA was isolated from the total RNA of the mycelium grown under cellulase induction conditions by oligo(dT)-cellulosse chromatography. The translation products in vitro were prepared by translating the isolated poly ($A^+$) RNA in rabbit reticulocyte lysate and analyzed by SDS-PAGE and fluorography. Of the translation products, CMCase I was identified by the immunoprecipitation against anti-CMCase I IgG.