• Asian-Australasian Journal of Animal Sciences
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• 제20권2호
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• pp.153-159
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• 2007

In our previous study, overexpression of extracellular proteinase inhibitor (Expi) gene accelerated apoptosis of mammary epithelial cells, and induced expression of B cell activating factor (BAFF) gene. In this study, we found induction of BAFF-receptor (BAFF-R) gene expression in the Expi-transfected cells. A proliferation-inducing ligand (APRIL) gene is another TNF family member and the closest known relative of BAFF. We found induction of APRIL gene expression in the Expi-overexpressed apoptotic cells. NF-${\kappa}$B gene was also induced in the Expi-overexpressed cells. Expression patterns of BAFF and APRIL pathway-related genes were examined in in vivo mouse mammary gland at various reproductive stages. Expression levels of BAFF gene were very low at early pregnancy, increased from mid-pregnancy, and peaked at lactation, and thereafter decreased at involution stages of mammary gland. Expression of BAFF-R gene was highly induced in involution stages compared to lactation stages. Thus, expression patterns of BAFF-R gene were correlated to apoptotic status of mammary gland: active apoptosis of mammary epithelial cells occurs at involution stage of mammary gland. Expression levels of NF-${\kappa}$B gene were higher in involution stages compared to lactation stages. We analyzed mRNA levels of bcl-2 family genes from different stages of mammary development. Bcl-2 gene expression was relatively constant during lactation and involution stages. There was a slight increase in bcl-xL gene expression in involution stages compared to lactation state. Bax gene expression was highly induced in involution stage. Our results suggest that signaling pathways activated by both BAFF and ARRIL in mammary gland point towards NF-${\kappa}$B activation which causes upregulation of bax.

• BMB Reports
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• 제40권5호
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• pp.678-683
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• 2007

Extracts from the leaves of the Ginkgo biloba are becoming increasingly popular as a treatment that is claimed to reduce atherosclerosis, coronary artery disease, and thrombosis. In this study, the effect of ginkgolide B (GB) from Ginkgo biloba leaves in collagen (10 ${\mu}g/ml$)-stimulated platelet aggregation was investigated. It has been known that human platelets release matrix metallo-proteinase-9 (MMP-9), and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GB to form an MMP-9 (86-kDa) on gelatinolytic activities. And then, activated MMP-9 by GB dose-dependently inhibited platelet aggregation, intracellular $Ca^{2+}$ mobilization, and thromboxane $A_2$ ($TXA_2$) formation in collagen-stimulated platelets. Activated MMP-9 by GB directly affects down-regulations of cyclooxygenase-1 (COX-1) or $TXA_2$ synthase in a cell free system. In addition, activated MMP-9 significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have the anti-platelet function in resting and collagen-stimulated platelets. Therefore, we suggest that activated MMP-9 by GB may increase the intracellular cAMP and cGMP production, inhibit the intracellular $Ca^{2+}$ mobilization and $TXA_2$ production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that activated MMP-9 is a potent inhibitor of collagen-stimulated platelet aggregation. It may act a crucial role as a negative regulator during platelet activation.

• Biomolecules & Therapeutics
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• 제18권3호
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• pp.300-307
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• 2010

Photosensitized peroxidation of membrane lipids has been implicated in skin pathologies such as phototoxicity and premature aging. We have previously reported that syriacusin compounds isolated from Hibiscus Syriacus inhibited lipid peroxidation. Here, we investigated whether syriacusins could be effective inhibitor to skin aging using ultraviolet-irradiated human dermal fibroblast cells (HDFCs). Syriacusins A, B, and C inhibit the activity of human neutrophil elastase (HNE), a serine protease to degrade extracellular matrix (ECM) proteins including elastin, with $IC_{50}s$ of 8.0, 5.2, and $6.1\;{\mu}M$, respectively. No changes in cell viability were detected by syriacusins A and B in UV-B ($10\;mJ/cm^2$) irradiated HDFCs. Matrix metallo-proteinase (MMP)-1 expression in HDFCs was increased by UV-B irradiation. MMP-1 expression in UV-B irradiated HDFCs was decreased by $10\;{\mu}M$ and $20\;{\mu}M$ syriacusin A to 50% and 20% of untreated control, respectively. Syriacusin B treated with $20\;{\mu}M$ reduced MMP-1 expression in UV-B irradiated HDFCs to 60% of untreated control. Syriacusin A also inhibited MMP-2 expression accompanying the increase of type-I pro-collagen in UV-B irradiated HDFCs. These results demonstrate that syriacusin A could be a more effective compound to inhibit skin aging caused by UV irradiation. It suggests that syriacusins A and B might be developed as possible agents to treat or prevent skin aging.

• Parasites, Hosts and Diseases
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• 제35권2호
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• pp.87-94
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• 1997

우리 나라에서 가장 감염자가 많은 기생충인 간흡충의 현 감염에 특이한 항원 분획을 찾고자 면역이적법을 이용하여 감염자의 혈청을 검색하였다. 실험적으로 토끼에서 얻은 간흡충 성충의 조항원에 시스테인계 단백질분해효소 억제제인 I-64를 첨가하였을 때 큰 분자량의 분획을 가장 잘 보존하여 200-14 kDa의 범위에 20개 이상의 분획을 관찰하였다. 이 조항원을 이용하여 경북 상주군 주민의 혈청을 면역이적법으로 검사하였다 대변검사, 피내반응검사, 효소면역법검사(ELISA)를 이 용하여 검사한 73명 중 충란양성자 49명의 혈청 내에 특이 IgG와 IgE 항체가 생성되었고, IgM과 IgA 항체는 특이하게 반응하지 않았다. 여러 항원 분획 중에서 43, 34, 28-25 kDa 항원이 현 감 염에 특이한 분획이고. 항체와 반응한 94, 80, 72, 68, 64, 62, 52, 47, and 40 kDa 분획은 특이하지 않은 분획임을 확인하였다. 추후에 각 항원과 이에 대한 혈청반응에 대하여 특성을 더 연구 할 필요가 있으며, 특히 치료후 추적검사를 통하여 혈청내 항체의 소실을 구명하여야 할 것이다.

• Plant Biotechnology Reports
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• 제5권1호
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• pp.27-34
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• 2011

Jasmonates control diverse plant developmental processes, such as seed germination, flower, fruit and seed development, senescence and tuberization in potato. To understand the role of methyl jasmonate (MeJA) in potato tuberization, the Arabidopsis JMT gene encoding jasmonic acid carboxyl methyltransferase was constitutively overexpressed in transgenic potato plants. Increases in tuber yield and size as well as in vitro tuberization frequency were observed in transgenic plants. These were correlated with JMT mRNA level-- the higher expression level, the higher the tuber yield and size. The levels of jasmonic acid (JA), MeJA and tuberonic acid (TA) were also higher than those in control plants. Transgenic plants also exhibited higher expression of jasmonate-responsive genes such as those for allene oxide cyclase (AOC) and proteinase inhibitor II (PINII). These results indicate that JMT overexpression induces jasmonate biosynthesis genes and thus JA and TA pools in transgenic potatoes. This results in enhanced tuber yield and size in transgenic potato plants.

• Plant Biotechnology Reports
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• 제5권3호
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• pp.217-224
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• 2011

Non-heading Chinese cabbage (Brassica rapa L. ssp. chinensis) is a popular vegetable in Asian countries. The diamondback moth (DBM), Plutella xylostella (L.), an insect with worldwide distribution, is a main pest of Brassicaceae crops and causes enormous crop losses. Transfer of the anti-insect gene into the plant genome by transgenic technology and subsequent breeding of insect-resistant varieties will be an effective approach to reducing the damage caused by this pest. We have produced transgenic non-heading Chinese cabbage plants expressing the potato proteinase inhibitor II gene (pinII) and tested the pest resistance of these transgenic plants. Non-heading Chinese cabbages grown for 45 days on which buds had formed were used as experimental materials for Agrobacterium-mediated vacuum infiltration transformation. Forty-one resistant plants were selected from 1166 g of seed harvested from the infiltrated plants based on the resistance of the young seedlings to the herbicide Basta. The transgenic traits were further confirmed by the Chlorophenol red test, PCR, and genomic Southern blotting. The results showed that the bar and pinII genes were co-integrated into the resistant plant genome. A bioassay of insect resistance in the second generation of individual lines of the transgenic plants showed that DBM larvae fed on transgenic leaves were severely stunted and had a higher mortality than those fed on the wild-type leaves.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1995년도 춘계학술대회
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• pp.114-114
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• 1995

이 연구에서는 혈전증 치료에 사용되는 혈전 응해제를 국내 독사독으로부터 개발하기 위한 실험을 실시하였다. 그 내용을 요약하면 다음과 같다. 국내에 서식하는 독사인 Agkistrodon blomoffi brevicaudus, Agkistrodon caliginosus와 Agkistrodon saxatilis에서 각각 사독을 채취하여 fibrin plate 방법으로 fibrin 분해능을 조사하여 Agkistrodon blomoffi brevicaudus의 독이 분해능이 가장 우수함을 밝혔다. 이와 같은 사실에 기초하여 A. blomoffi brevicaudus의 독으로부터 p-Aminobenzamidine affinity chromatography와 DEAE ion-exchange chromatography를 이용하여 분자량이 50.8 kDa인 황성 단백질을 정제하였다. 위에서와 같은 방법으로 정제한 단백질은 fibrin 분해능이 우수하고 fibrinogen의 ${\gamma}$ chain은 분해하지 않으나 B$\beta$ chain을 $A\alpha$ chain에 비하여 보다 선택적으로 분해하는 단백분해 효소임을 증명하였다. 이 정제 효소의 Fibrin에 대한 분해능은 266$\mu\textrm{g}$/${\mu}\ell$의 농도에서 Plasmin 1.0 unit (=3.0 WHO unit)보다 높게 나타났다. 정제된 효소는 chromogenic substrate인 N-Benzoyl-Phe-Val-Arg-pNA와 N-p-Tosyl- Gly-Pro-Arg-pNA의 arginine carboxyl side를 분해하고 pH 7.5에서 최대 활성을 보이며, Vmax는 5.46 umo1/1ㆍmin이고, Km 값은 0.20mM이며, 그리고 Cu$^{2+}$, $Zn^{2+}$, soybean trypsin inhibtor에 의해 25～50% 정도, serine proteinase inhibitor인 phenylmethylsulfonyl floride에 의해 80%정도 활성이 억제되는 특성이 있음을 규명하였다.

• Archives of Pharmacal Research
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• 제28권7호
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• pp.816-822
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• 2005

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

• Current Research on Agriculture and Life Sciences
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• 제32권3호
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• pp.149-154
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• 2014

1. Pepsin과 pancreatin 소화물들을 비교한 결과, 순수한 팥 단백질 중에서 소화효소 저항성을 가지는 단백질이 존재하는 것으로 확인되었다. 2. 팥의 주요 단백질을 제거하고 pepsin과 pancreatin으로 소화시켰을 때 더 많은 분해가 일어나는 것으로 보아 팥의 주요단백질들 중에 소화효소 저항성을 가지는 것이 많을 것이라 추측된다. 3. 팥의 주요 단백질들은 장 점막세포와는 크게 작용하지 않는 것을 확인할 수 있었다. 4. 팥 단백질의 데이터베이스 구축과 팥 단백질이 다른 영양소들과의 상호작용을 하는 지에 대한 연구가 진행되어야 할 것이다.

• 한국발생생물학회지:발생과생식
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• 제5권1호
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• pp.23-33
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• 2001

포유류의 난자가 수란관내로 배란될 때는 난포액 성분도 같이 수란관내로 들어간다. 본 연구에서는 처음으로 난포액의 일부 성분이 수란관액에 의해서 변화하는 것을 관찰하였다. 사람의 난포액을 gelatin zymogram으로 분석한 결과 621kDa gelatinase 이외에 110kDa gelatinase (GA110) 등의 여러 gelatinase 활성이 나타났다. 이 활성들은 EDTA나 phenanthroline에 의해 억제된 반면 PMSF 처리에 의해서는 아무런 변화가 없었다. 소의 수란관액에서는 62kDa gelatinase의 활성만이 주로 관찰되었다. 소의 수란관 액과 사람의 난포액을 1:1로 섞고 이를 37$^{\circ}$C에서 3시간 동안 둔 결과 사람의 난포액의 GA110 활성은 사라졌다. 사람의 난포액에 APMA를 첨가한 결과 GA110의 활성은 대부분 감소하고 대신 62kDa gelatinase의 활성은 오히려 증가하였다. 반면에 사람의 난포액에 EDTA를 3시간 동안 처리한 결과 GA110의 활성은 오히려 현저히 증가하였고 이 때 다른 gelatinases의 활성은 영향을 받지 않았다. PMSF나SBTI는 난포액내의 gelatinases활성에 아무런 변화를 일으키지 않았다. EDTA, PMTA 혹은 SBTI 등의 proteinase inhibitor를 미리 처리한 사람의 난포액에 소의 수란관 내액을 섞은 경우에도GA110의 활성은 여전히 감소하였다. 사람의 혈청에서도 EDTA에 의해 활성이 현저히 증가하는 GA110이 발견되었다. 사람의 난포액과 유사하게 혈청내의 GA110도 소의 수란관액에 의하여 활성이 사라졌다. 그러나 사람의 난포과립세포의 추출물에서는 단지 92kDa gelatinase만 관찰이 되었다. 마지막으로 anti-human gelatinase A 항체를 사용하여 사람의 난포액과 혈청 그리고 난포과립세포의 추출액을 western blotting한 결과 621kDa과 GA110 만이 항원-항체 반응을 나타내었다. 이 같은 결과로 미루어 사람의 난포액과 혈청에는 gelatinase A의 독특한 isoform인 GA110이 있으며 특히 난포액내의 GA110은 수란관액성분에 의해 선택적으로 분해되는 것으로 여겨진다.