• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.445-450
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• 1996

Gelatinase A is a member of the matrix metalloproteinases that play an important role in cancer invasion and metastasis. In the course of screening gelatinase A inhibitors from microbial sources, a fungal strain PT-262 showed a strong inhibitory activity. The strain was identified as Chaunopycnis alba on the basis of its morphological characteristics. The inhibitor was isolated from acetone extract of mycelial cake by sequential chromatographies on MCI-gel, Sephadex LH-20, and a reverse-phase HPLC column. The purified inhibitor was identified as pyridoxatin by its physico-chemical properties and spectroscopic analysis. Pyridoxatin is not a peptide analog and has cyclic hydroxamic acid moiety. It inhibited activated gelatinase A with an $IC_{50}$ value of 15.2 ${\mu}M$ using fluorescent synthetic peptide. It also had a strong cytotoxicity against human cancer cell lines in vitro. Furthermore, this compound inhibited DNA synthesis with an $IC_{50}$ value of 2.92 ${\mu}M$ in PC-3 prostate cancer cells by [$^3H$]thymidine incorporation assay.

• Journal of Microbiology and Biotechnology
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• 제28권12호
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• pp.2046-2056
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• 2018

Phospholipase D has great commercial value due to its transphosphatidylation products that can be used in the food and medicine industries. In order to construct a strain for use in the production of PLD, we employed a series of combinatorial strategies to increase PLD expression in Bacillus subtilis WB600. These strategies included screening of signal peptides, selection of different plasmids, and optimization of the sequences of the ribosome-binding site (RBS) and the spacer region. We found that using the signal peptide amyE results in the highest extracellular PLD activity (11.3 U/ml) and in a PLD expression level 5.27-fold higher than when the endogenous signal peptide is used. Furthermore, the strain harboring the recombinant expression plasmid pMA0911-PLD-amyE-his produced PLD with activity enhanced by 69.03% (19.1 U/ml). We then used the online tool \RBS Calculator v2.0 to optimize the sequences of the RBS and the spacer. Using the optimized sequences resulted in an increase in the enzyme activity by about 26.7% (24.2 U/ml). In addition, we found through a transfer experiment that the retention rate of the recombinant plasmid after 5 generations was still 100%. The final product, phosphatidylserine (PS), was successfully detected, with transphosphatidylation selectivity at 74.6%. This is similar to the values for the original producer.

• 동의생리병리학회지
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• 제30권1호
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• pp.40-46
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• 2016

Many medicinal plants have been used for the treatment of edema, jaundice, and gonorrhea in traditional Oriental medicine. This screening study was designed to search the positive inotropic effects of herbal extracts in beating rabbit atria. Aquarius extracts of twenty six herbs were examined in atrial mechanical dynamics such as pulse pressure and stroke volume and atrial natriuretic peptide (ANP), one of the main hormones involved in the regulation of the body fluid and blood pressure homeostasis in perfused beating rabbit atria. Sophora flavescens Ait., Rheum officinale Baill., Acorus gramineus Sol., Chelidonium majus L., Pulsatilla koreana Nakai., Reynoutria japonica Houtt., Euphorbia lathyris L., Pyrrosia lingua (Thunb.) Farwell, Poncirus trifoliata Rafin., Anemarrhena asphodeloides Bunge, Kochia scoparia Schrad. significantly increased stroke volume and pulse pressure. However, those herbal extracts were not induced ANP secretion. We clarified the eleven herbal extracts for the positive inotropic effect independent of ANP secretion in beating rabbit atria. Thus these results provide a beneficial data for the treatment of the impairment of body fluid and blood pressure in traditional Oriental medicine.

• 한국식품과학회지
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• 제28권4호
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• pp.684-688
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• 1996

전통식품인 된장으로부터 혈소판 응집 저해활성을 갖는 항혈전 펩타이드를 탐색하고자 하였다. 증류수로 추출한 된장액을 한외 여과하여 얻은 분자량 3,000 dalton 이하의 여액에 대하여, SD흰쥐의 세척혈소판 (washed platelet)을 이용하여 ADP 자극으로 유도되는 혈소판응집의 저해정도를 aggregometer에서의 탁도로 측정하였을 때, $96\;{\mu}g/ml$로 처리시 90% 가량의 응집 저해효과가 확인되었다. 추출물을 Dowex 50W X-2 강양이온 교환칼럼을 이용하여 pyridine-formic acid 완충액의 pH (2.8-5.0) 및 몰농도(0.1-3.0M)를 단계별로 증가시키면서 용출시켜 19개의 획분(No.B-18)으로 분리하였다. 이들 획분의 활성을 microplate를 이용한 방법으로 측정하였을 때, 전체적으로 앞의 방법보다는 감도가 낮게 나타났으나 모든 분획에서 응집억제 효과가 확인되었다. 이때 용량반응 곡선으로부터 구한 각 획분의 $IC_{50}$은 $10-1,000\;{\mu}g/ml)$의 범위를 보였으며, 대부분의 획분들은 양성대조구인 RGDS $(IC_{50},\;205\;{\mu}g/ml)$보다 높은 활성을 나타내었다. 또한, 후반부의 염기성 획분$(IC_{50},\;10-20\;{\mu}g/ml)$에서 된장추출물$(IC_{50},\;30\;{\mu}g/ml)$보다 활성이 높게 나타났다. 이들 중 활성이 가장 높은 16번 획분을 아미노산 분석한 결과, 펩타이드 부분에 histidine, arginine, alanine 등이 높은 비율로 존재하였다.

• IMMUNE NETWORK
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• 제21권5호
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• pp.34.1-34.16
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• 2021

Sjögren's syndrome (SS) is an autoimmune disease characterized by dryness of the mouth and eyes. The glandular dysfunction in SS involves not only T cell-mediated destruction of the glands but also autoantibodies against the type 3 muscarinic acetylcholine receptor or aquaporin 5 (AQP5) that interfere with the secretion process. Studies on the breakage of tolerance and induction of autoantibodies to these autoantigens could benefit SS patients. To break tolerance, we utilized a PmE-L peptide derived from the AQP5-homologous aquaporin of Prevotella melaninogenica (PmAqp) that contained both a B cell "E" epitope and a T cell epitope. Repeated subcutaneous immunization of C57BL/6 mice with the PmE-L peptide efficiently induced the production of Abs against the "E" epitope of mouse/human AQP5 (AQP5E), and we aimed to characterize the antigen specificity, the sequences of AQP5E-specific B cell receptors, and salivary gland phenotypes of these mice. Sera containing anti-AQP5E IgG not only stained mouse Aqp5 expressed in the submandibular glands but also detected PmApq and PmE-L by immunoblotting, suggesting molecular mimicry. Characterization of the AQP5E-specific autoantibodies selected from the screening of phage display Ab libraries and mapping of the B cell receptor repertoires revealed that the AQP5E-specific B cells acquired the ability to bind to the Ag through cumulative somatic hypermutation. Importantly, animals with anti-AQP5E Abs had decreased salivary flow rates without immune cell infiltration into the salivary glands. This model will be useful for investigating the role of anti-AQP5 autoantibodies in glandular dysfunction in SS and testing new therapeutics targeting autoantibody production.

• BMB Reports
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• 제44권9호
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• pp.607-612
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• 2011

Several investigators have shown that CpG-DNA has outstanding effects as a Th1-responsive adjuvant and that its potent adjuvant effects are enhanced by encapsulation with a liposome of proper composition. In this study, we showed that encapsulation with phosphatidyl-${\beta}$-oleoyl-${\gamma}$-palmitoyl ethanolamine (DOPE): cholesterol hemisuccinate (CHEMS) complex enhances the immunostimulatory activity of CpG DNA and the binding of CpG-DNA to TLR9. We also examined involvement of myeloid differentiation protein (MyD88) and NF-${\kappa}B$ activation in liposome-encapsulated CpG-DNA-induced IL-8 promoter activation. In this manuscript, the natural phosphodiester bond CpG-DNA encapsulated by DOPE : CHEMS complex is designated as Lipoplex(O). Importantly, we successfully screened B cell epitopes of envelope protein (E protein) of hepatitis C virus (HCV-E) and attachment glycoprotein G of human respiratory syncytial virus (HRSV-G) by immunization with complexes of several peptides and Lipoplex(O) without carriers. Therefore, Lipoplex(O) is potentially applicable as a universal adjuvant for peptide-based epitope screening and antibody production.

• BMB Reports
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• 제28권1호
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• pp.83-86
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• 1995

In the course of screening for new aminopeptidase M inhibitors which were expected to be analgesic, immunopotentiating, or anti-metastatic agents, the novel synthetic substance MR-387C[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl-L-prolyl-L-leucine] (M.W. 504 daltons) was obtained. It was competitive with the substrate and had an $IC_{50}$ value of $0.04\;{\mu}m/ml$ ($7.9{\times}10^{-8}\;M$) and an inhibition constant ($K_i$) of $3.8{\times}10^{-8}\;M$. This novel MR-387C was compared with various known inhibitors of aminopeptidase M. It inhibited the enzyme more strongly than any other microorganism-originated inhibitor, except probestin.

• 약학회지
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• 제55권3호
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• pp.203-212
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• 2011

Adipocytes have been known to secrete a number of important proteins called adipokines with roles in energy metabolism, reproduction, cardiovascular function and immunity. In this study we have attempted to identify intensively secretory proteins from 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated into mature adipocytes and then the cells were left in serum-free medium. The supernatant was filtrated and dialyzed. Lyophilized secretome was fractionated by two different methods, 1-D SDS PAGE and RP-FPLC. The tryptic peptides from the gel slices and the FPLC fractions were analyzed by nanoLC/ESI-MS/MS. We identified a total of 303 identical proteins from two methods, 251 proteins from 1-D gel and 184 proteins from RP-FPLC. 86 of them were listed as a secretory protein Finally, we identified many known or unknown secreted proteins existed in the low level including adiponectin, angiotensinogen, bone morphogenetic protein-1 (BMP-1), macrophage migration inhibitory factor (MIF), insulin like growth factor-II (IGF-II), interleukin-6 (IL-6), follistatin-related protein-1, minecan, and resistin. The existence of some of secreted proteins has been confirmed in RNA level. This proteomic experiment is useful for the intensive screening of secretory proteins in many kinds of other cells.

• Bulletin of the Korean Chemical Society
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• 제30권3호
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• pp.577-581
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• 2009

Interactions between the surface and capsid proteins of the hepatitis B virus (HBV) are critical for the assembly of virus particles. In this study, we developed a cell-based method to visualize the interactions between the capsid and surface proteins of HBV. Capsid-GFP, a capsid protein fused to a green fluorescence protein (GFP), forms nucleocapsid-like structures in the cytoplasm of mammalian cells. It relocates to the plasma membranes in cells expressing PH-PreS, a fusion protein consisting of the PreS region of the HBV surface protein and the PH domain of PLC-$\gamma$. Membrane localization of the capsid-GFP in these cells is prevented by an inhibitory peptide that blocks the interaction between the capsid and surface proteins. This dynamic localization of capsid-GFP is applicable for screening compounds that may potentially inhibit or prevent the assembly process of HBV particles.

• 생명과학회지
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• 제10권2호
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• pp.218-227
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• 2000

Bone morphogenetic protein 1 (BMP-1) is part of a complex capable of inducing ectopic bone formation in mammals. Studies on TGF-β1 processing and Drosophila dorsal-ventral patterning have focused attention on BMP-1 as important in mediating the biological activity of this bone inducing complex. Herein, the bacterial expression, refolding, purification, and initial characterization of the BMP-1 proteolytic domain (BPD) are described. A semi-quantitative fluorescence-based thin layer chromatography assay was developed to assist in rapidly screening for optimal renaturation conditions. According to a preliminary screen for optimal conditions for the refolding of BPD , a detectable proteolytic activity against a high turnover substrate for astacin, a homologous protease from crayfish was observed. The conditions identified have allowed the expression of sufficient amounts of BPD for the characterization of the protein. Its proteolytic activity exhibits the same cleavage specificity as astacin against seven substrates that were previously synthesized for studying astacin. Furthermore, this activity is inhibited by the metal chelator 1,10-phenanthroline but not by its analogue 1,7-phenanthroline. The collagenase inhibitor Pro-Leu-Gly hydroxamate was found to inhibit both astacin and BPD activity. The results presented in this paper argue that BMP-1 does in fact possess an intrinsic proteolytic activity.