• 대한한의학회지
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• 제25권3호
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• pp.123-136
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• 2004

Objectives : The purpose of this investigation is to evaluate the effects of Woohwangcheongsim-won (WC) on the in vitro neuronal development and alteration in gene expression in a hypoxia model using cultured rat cortical cells. Methods : E/sub 18/ rat cortical cells were grown in a neurobasal medium containing B27 supplement and various concentration of WC. Initial development of growth cone was investigated by phase-contrast microscopy, while dendritic spine formation and synaptogenesis were investigated by immunocytochemistry with SynGAPα(a postsynaptic marker) and synaptophysin (presynaptic marker) antibodies. Alteration in gene expression was analyses by microarray using rat 5K-TwinChips. Results : WC suppressed the development of growth cones and WC increased the number of dendritic spines at 20 and 50㎍/mL concentration but there was no statistical significance. Instead, it significantly decreased the number at 100㎍/mL. The expression of anti-apoptosis gene Bcl2-like 1 (Bcl211) increased (Global M=0.46), while Akt1 decreased. Proapoptosis genes Bad and PDCD2 increased. The expression of hemoglobin alpha 1 (probably neuroglobin) increased (Global M=0.93). The expression of antioxidants such as catalase, heme oxygenase (HO), and PRKAG2 gene increased. The expression PKC gene increased. The expression of retinoic acid receptor alpha (RARα) increased significantly (Global M=1.0). Conclusions : These data suggest that WC trends to suppress cellular activity slightly in normoxia and increases the expression of apoptosis-, antioxidation-, oxygen capture-related genes in hypoxia, but increases Bcl111 that anti-apoptosis gene, on the other hand increases Bad, PDCD2 that pro-apoptosis genes, too..

• 한국수산과학회지
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• 제31권6호
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• pp.852-858
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• 1998

1996년 7월 마산만의 해수를 0.8 $\mu$m filter에 여과한 여과액과 f/2배지에서 배양한 P. micans와의 혼합배양액에서 P. micans를 사멸시키는 해양세균을 분리하였다. 분리균의 형태학적 및 생화학적 검사와 균체지방산을 분석하여 동정한 결과 Pseudomonas 속으로 동정되어 Pseudomonas sp. LG-2로 명명하였으며, 그 살조특성을 조사한 결과는 아래와 같다. Pseudomonas sp. LG-2의 개체수와 배양여과액의 농도가 높을수록 P. micans의 사멸효과가 높게 나타났다. $1.3\times10^5,\;1.3\times10^6$ cells/ml의 농도로 접종한 시험관의 P. micans는 급격히 감소하여 배양 7일 후에는 $10^2$ cells/ml 이하로 사멸되었다. 또한, 배양석과액의 최종농도가 $30\%$일 경우에는 급격히 감소하여 배양 3일 후 거의 사멸하였다. Pseudomonas sp. LG-2의 성장시기에 따른 배양여과액의 사멸효과는 잠복기의 경우 P. micans의 성장에 큰 영향을 미치지 않았으나, 대수증식기 중기의 배양여과액은 접종 5일 후 P. micans의 개체수를 1/2로 감소시켰으며, 정상기의 배양여과액은 접종 후 급격히 감소시켜 배양 3일 후 대부분 사멸시켰다. Alexandrium tamarense, Prorocentrum micans, Scrippsiella trochoidea, Gymnodinium sanguineum, Cochlodinium polykrikoides의 5종의 적조원인 편모조류에 대한 Pseudomonas sp. LG-2의 살조특이성은 P. micans와 S. trochoidea에 살조효과를 나타내었다.

• Journal of Periodontal and Implant Science
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• 제30권2호
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• pp.347-360
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• 2000

Bone remodeling results from the combined process of bone resorption and new bone formation which is regulated in part by some of the polypeptide growth factors such as platelet derived growth factor(PDGF), which has been known to be an important local regulator of bone cell activity and participate in normal bone remodeling. This process includes strictly regulated gene expression of several bone matrix proteins such as type I collagen and osteopontin, a 44 kDa phosphorylated glycoprotein, which has important roles in bone formation. The purpose of this study is to evaluate the effecs of PDGF-BB on the mRNA expression of bone matrix protein, type I collagen and osteopontin, in MC3T3- E1 cell culture. Cells were seeded at $5{\times}10^5$ cells in 10 ml of minimum essential medium alpha(${\alpha}-MEM$) containig 10% fetal bovine serum, 10 mM beta glycerophosphate. 0.1, 1, 10 ng/ml PDGF-BB were added to the cells for the day 3, 7, 14, 21, 28 and cultured for 24 hours. Type I collagen cDNA, Hf677, and osteopontin cDNA were used as probes for northern blot analysis. Total cellular RNA was purified at indicated day and northern blot analysis was performed. The results were as follows : Type I collagen mRNA expressions were higher at the day 3 and 7, and lower in the day 14, 21 in the control groups. In the experimental groups, mRNA expressions were increased when 0.1 ng/ml PDGF-BB were added on the day 3, 7, 21, and decreased in dose-dependent manner on the day 14, decreased at all added dose on the day 28. Osteopontin mRNA expressions were highest in the day 21 groups and lowest in the day 14 groups in the control groups. Interesting results were shown in the day 14 and 21 groups. We found that osteopontin mRNA level was increased in dose dependent manner in the day 14 groups, and decreased dose dependent manner in the day 21 groups. In conclusion, PDGF-BB may have various control effects on type I mRNA expression in the growth and differentiation process of MC3T3-E1 cells and may have contrary regulatory effects on osteopontin mRNA expression. For examples, when the baseline level of osteopontin mRNA was low, as in the day 14, PDGF-BB up-regulated osteopontin mRNA expression in dose dependent manner, and when the baseline level was high as in the day 21, PDGF-BB down-regulated dose dependent manner. Thus, it may be useful for clinical application in periodontal regeneration procedure if further study were performed.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.81-89
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• 2004

Tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and lymphotoxin-$\alpha$ (LT-$\alpha$, TNF-$\beta$) can initiate and perpetuate human diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and insulin-dependent diabetes mellitus (IDDM). TNFs can be blocked by the use of soluble TNF receptors. However, since monomeric soluble receptors generally exhibit low affinity or function as agonists, the use of monomeric soluble receptors has been limited in the case of cytokines such as TNF-$\alpha$, TNF-$\alpha$, interleukin (IL)-1, IL-4, IL-6, and IL-13, which have adapted to a multi component receptor system. For these reasons, very high-affinity inhibitors were created for the purpose of a TNFs antagonist to bind the TNFR and trigger cellular signal by using the multistep polymerase chain reaction method. First, recombinant simple TNFR-Ig fusion proteins were constructed from the cDNA sequences encoding the extracellular domain of the human p55 TNFR (CD120a) and the human p75 TNFR (CD120b), which were linked to hinge and constant regions of human $IgG_1$ heavy chain, respectively using complementary primers (CP) encoding the complementary sequences. Then, concatameric TNFR-Ig fusion proteins were constructed using recombinant PCR and a complementary primer base of recombinant simple TNFR-Ig fusion proteins. For high level expression of recombinant fusion proteins, Chinese hamster ovary (CHO) cells were used with a retroviral expression system. The transfected cells produced the simple concatameric TNFR-Ig fusion proteins capable of binding TNF and inactivating it. These soluble versions of simple concantameric TNFR-Ig fusion proteins gave rise to multiple forms such as simple dimers and concatameric homodimers. Simple TNFR-1g fusion proteins were shown to have much more reduced TNF inhibitory activity than concatameric TNFR-Ig fusion proteins. Concatameric TNFR-Ig fusion proteins showed higher affinity than simple TNFR-Ig fusion proteins in a receptor inhibitor binding assay (RIBA). Additionally, concatameric TNFR-Ig fusion proteins were shown to have a progressive effect as a TNF inhibitor compared to the simple TNFR-Ig fusion proteins and conventional TNFR-Fc in cytotoxicity assays, and showed the same results for collagen induced arthritis (CIA) in mice in vivo.

• Applied Microscopy
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• 제40권4호
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• pp.201-209
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• 2010

한방에서 사용되는 약재 중 가자와 지모, 단삼은 면역조절 및 항암효과, 항염증효과가 있다고 알려졌다. 그러나 비만세포와 관련된 아나필락스를 이해할 수 있는 세포학적 기전에 대한 가자와 지모, 단삼의 효과에 대해서는 알려진 바 없다. 본 연구에서는 가자와 지모, 단삼의 메탄올 추출물이 아나필락시스 반응에 대해 어떠한 영향을 끼치는지를 조사하였다. 시험관내 실험과 생체 실험을 통하여, 가자와 지모, 단삼의 메탄올 추출물이 compound 48/80에 의한 아나필락시스와 비만세포 활성화 및 IgE에 의한 피부반응을 관찰하였다. 실험결과는 다음과 같다. 1) 가자와 지모, 단삼의 메탄올 추출물은 compound 48/80에 의한 전신성 아나필락시스와 귀부종 반응, IgE에 의한 피부반응을 억제하였다. 2) Compound 48/80에 의한 비만세포 탈과립과 흰쥐 복강 비만세포로부터 히스타민 유리가 가자와 지모, 단삼의 메탄올 추출물 전처리에 의해 억제되었다. 3) Compound 48/80에 의한 흰쥐 복강 비만세포내로의 칼슘 유입이 가자와 지모, 단삼의 메탄올 추출물 전처리에 의해 현저하게 억제되었다. 이상의 결과로 가자와 지모, 단삼의 메탄올 추출물은 비만세포 탈과립과 비만세포로부터 히스타민 유리, 비만세포내로 칼슘유입을 억제함으로써 compound 48/80에 의한 아나필락시스와 IgE에 의한 피부반응을 억제한다는 것을 알 수 있었다. 이로써 가자와 지모, 단삼은 알레르기 질환에 대한 효과적인 치료제로 이용될 수 있을 것으로 생각된다.

• 생명과학회지
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• 제22권8호
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• pp.1018-1023
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• 2012

Snail은 E-cadherin 발현을 직접 억제하는 zinc finger transcription factor로서, 암세포의 invasion과 metastasis를 촉진시키는 epithelial-mesenchymal transition (EMT)를 유발한다. 또한 Snail은 세포사멸 자극과 세포 생존물질의 제거로 인한 세포사멸에 대해 저항성을 나타낸다. 그러나 이에 대한 분자기작은 잘 알려져 있지 않다. 본 연구에서는 가장 널리 사용되는 항암제 중의 하나인 5-fluorouracil (5-FU)에 의한 세포사멸에 대한 Snail의 저항성 기작에 대하여 조사하였다. MCF-7 #5 세포주에 doxycycline (DOX)을 처리하여 Snail을 과발현시킨 세포에서 5-FU에 의한 세포사멸이 억제되고 세포괴사가 일어남을 확인하였다. DOX 처리 및 Snail expression vectors인 pCR3.1-Snail-Flg와 phosphorylation-resistant mutant Snail vector인 pCR3.1-S104, 107A Snail-Flg을 이용하여 Snail을 과발현 시킨 경우 ERK1/2의 활성에는 영향을 주지 않는 반면 PTEN 발현억제 및 불활성화, 그리고 Akt/PKB 활성화가 유도됨을 관찰하였다. 또한, Snail은 5-FU에 의한 p53의 발현을 억제한다는 사실을 확인하였다. 따라서 Snail은 prosurvival kinase인 Akt/PKB의 활성화와 p53 억제를 통해 5-FU에 의한 세포사멸을 세포괴사로 전환하는 것으로 생각된다.

• 생명과학회지
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• 제19권1호
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• pp.129-137
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• 2009

$Ca^{2+}$은 다양한 자극과 빛, biotic, abiotic 스트레스, 호르몬 등의 반응에 대한 세포내 2차 신호전달물질로써 중요한 역할을 한다. $Ca^{2+}$의 반응자들은 특정 물질과 경로를 활성화함으로써 신호전달 기능을 한다고 알려져 있는 $Ca^{2+}$ 결합 단백질들이다. 이들 단백질 중, calmidulin (CaM)은 식물과 동물의 특정 단백질의 활성을 조절하는 것으로 잘 알려져 왔다. 특히, 식물은 다양한 CaM 유전자와 특징적인 protein kinase와 전사인자를 포함한 많은 종류의 CaM 관련 단백질들을 가지고 있다. 이로 인해서 식물은 주변의 여러 가지 신호등을 인지할 수 있을 뿐만 아니라 변화된 환경에 적응할 수 있는 것이다. 하지만, 대부분의 CaM이나 이들과 관련된 단백질들의 기능은 최근 활발히 연구되고 있지만 아직 많은 작용 기작이 연구의 대상이 되고 있다. 따라서 CaM의 기능을 좀 더 이해한다면 식물의 환경적 자극에 대한 반응과 식물의 성장과 발달에 있어서 CaM의 역할을 규명하는데 도움을 줄 수 있을 것으로 기대된다. 본 논문은 $Ca^{2+}$-CaM의 신호전달 시스템과, CaM과 관련된 단백질들, 그리고 식물의 biotic, abiotic 스트레스에 대한 외부 자극의 반응에 있어서 CaM의 작용에 대해 기술하였다.

• Molecules and Cells
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• 제38권5호
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• pp.409-415
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• 2015

Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. ${\Delta}^5$-3-ketosteroi isomerase (KSI) catalyzes the allylic isomerization of ${\Delta}^5$-3-ketosteroid to its conjugated ${\Delta}^4$-isomers at a rate that approache the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 $O{\eta}$ and C3-O of equilenin an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 $O{\eta}$ and C3-O of the bound steroi was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1-11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7-2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.

• Molecules and Cells
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• 제39권12호
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• pp.855-861
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• 2016

Ginsenosides, which are the active materials of ginseng, have biological functions that include anti-osteoporotic effects. Aqueous ginseng extract inhibits osteoclast differentiation induced by receptor activator of NF-${\kappa}B$ ligand (RANKL). Aqueous ginseng extract produces chromatography peaks characteristic of ginsenosides. Among these peaks, ginsenoside Re is a major component. However, the preventive effects of ginsenoside Re against osteoclast differentiation are not known. We studied the effect of ginsenoside Re on osteoclast differentiation, RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity, and formation of multinucleated osteoclasts in vitro. Ginsenoside Re hampered osteoclast differentiation in a dose-dependent manner. In an in vivo zebrafish model, aqueous ginseng extract and ginsenoside Re had anti-osteoclastogenesis effects. These findings suggest that both aqueous ginseng extract and ginsenoside Re prevent bone resorption by inhibiting osteoclast differentiation. Ginsenoside Re could be important for promoting bone health.

• 생명과학회지
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• 제16권5호
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• pp.716-722
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• 2006

Superoxide dismutase (SOD) is physiologically important in regulating cellular homeostasis and apoptotic cell death, and its mutations are the cause of familial amyotrophic lateral sclerosis (FALS). Mitochondrial serine protease HtrA2 has a pro-apoptotic function and has known to be associated with neurodegenerative disorders. To investigate the relationship between genes associated with apoptotic cell death, such as HtrA2 and SOD1, we utilized the pGEX expression system to develop a simple and rapid method for purifying wild-type and ALS-associated mutant SOD1 proteins in a suitable form for biochemical studies. We purified SOD1 and SOD1 (G93A) proteins to approximately 90% purity with relatively high yields (3 mg per liter of culture). Consistent with the result in mammalian cells, SOD1 (G93A) was more insoluble than wild-type SOD1 in E. coli, indicating that research on the aggregate formation of SOD1 may be possible using this pGEX expression system in E. coli. We investigated the HtrA2 serine protease activity on SOD1 to assess the relationship between two proteins. Not only wild-type SOD1 but also ALS-associated mutant SOD1 (G93A) were cleaved by HtrA2, resulting in the production of the 19 kDa and 21 kDa fragments that were specific for anti-SOD1 antibody. Using protein gel electrophoresis and immunoblot assay, we compared the relative molecular masses of thrombin-cleaved GST-SOD1 and HtrA2-cleaved SOD1 fragments and can predict that the HtrA2-cleavage sites within SOD1 are the peptide bonds between leucine 9-lysine 10 (L9-K10) and glutamine 23-lysine 24 (Q23-K24). Our study indicates that SOD1 is one of the substrate for HtrA2, suggesting that both HtrA2 and SOD1 may be important for modulating the HtrA2-SOD1-mediated apopotic cell death that is associated with the pathogenesis of neurodegenerative disorder.