• KSBB Journal
• /
• 제10권2호
• /
• pp.131-136
• /
• 1995

본 실험에서는 인간 전과립 세포를 회분배양 조건 에서 10%의 혈청을 포함하는 배지로 배양하였다. 이 와같은 조건에서 세포의 비성장율은 0.374C1/day) 이고 세포내 존재하는 살균활성을 가진 목적 단백질 의 생산성은 0.435(mg/10' viable cells)으로 나타났 다. 세포내 존재하는 목척 단백질의 순수 분리를 위 해 sephadex G-75, sephadex G-100, Bio→Rex70 을 이용해 분리하였다. 정제된 목적 단백질은 감수성 균주, E. coli IFO 13168과 St. aureus IFO 3060 을 표준 균주로 살균 측정을 하였을 때, 기존의 항생 물질은 균주에 대해 40~701땅1m!에서 활성을 나타 낸 반면, 정제된 목적 단백질은 G(+)균(St. aureus IFO 3060)에서는 $0.5\mu\textrm{g}$/ml이고, G(-)균(E. coli IFO 13168)에서는 $0.4\mu\textrm{g}$/ml에서 살균 활성을 보였다. 따라서 AMF가 기존 항생물질보다 살균 활성이 강력한 단백질임을 알 수 있다. 해당 분자량을 측정 하기 위해 SDS-PAGE로 측정한 결과 약 15,000 Dalton의 분자량을 갖고 있음이 확인됐다.

• 대한본초학회지
• /
• 제20권3호
• /
• pp.29-41
• /
• 2005

Objectives : The use of natural products with therapeutic properties is as ancient as human civilisation and, for a long time, mineral, plant and animal products were the main sources of drugs. Catalposide, the major iridoid glycoside isolated from the stem bark of Catalpa ovata G. Don (Bignoniceae) has been shown to possess anti-microbial and anti-tumoral properties. Heme oxygenase-1 (HO-1) is a stress response protein and is known to play a protective role against the oxidative injury. In this study, we examined whether catalposide could protect Neuro 2A cells, a kind of neuronal cell lines, from oxidative damage through the induction of HO-1 protein expression and HO activity. We also examined the effects of catalposide on the productions of tumor necrosis $factor-{\alpha}\;(TNF-{\alpha})$ and nitric oxide (NO) on RAW 264.7 macrophages activated with the endotoxin lipopolysaccharide. Methods : HO-1 expression in Neuro 2A cells was measured by Western blotting analysis. NO and $TNF--{\alpha}$ produced by RAW 264.7 macrophage were measured by Griess reagent and enzyme-linked immunosorbent assay, respectively. Results : The treatment of the cells with catalposide resulted in dose- and time-dependent up-regulations of both HO-1 protein expression and HO activity. Catalposide protected the cells from hydrogen peroxide-induced cell death. The protective effect of catalposide on hydrogen peroxide-induced cell death was abrogated by zinc protoporphyrin IX, a HO inhibitor. Additional experiments revealed the involvement of CO in the cytoprotective effect of catalposide-induced HO-1. In addition, catalposide inhibited the productions of $TNF--{\alpha}$ and NO with significant decreases in mRNA levels of $TNF--{\alpha}$ and inducible NO synthase. Conclusions : Our results indicate that catalposide is a potent inducer of HO-1 and HO-1 induction is responsible for the catalposide-mediated cytoprotection against oxidative damage and that catalposide may have therapeutic potential in the control of inflammatory disorders.

• Journal of Microbiology and Biotechnology
• /
• 제16권9호
• /
• pp.1429-1433
• /
• 2006

Application of recombinant protein production and particularly their isotopic enrichment has stimulated development of a range of novel multidimensional heteronuclear NMR techniques. Peptides in most cases are amenable to assignment and structure determination without the need for isotopic labeling. However, there are many cases where the availability of $^{15}N$ and/or $^{13}C$ labeled peptides is useful to study the structure of peptides with more than 30 residues and the interaction between peptides and membrane. CRAMP (Cathelicidin-Related AntiMicrobial Peptide) was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptides. CRAMP was successfully expressed as a GST-fused form in E. coli and purified using affinity chromatography and reverse-phase chromatography. The yield of the CRAMP was 1.5 mg/l 1. According to CD spectra, CRAMP adopted ${\alpha}$-helical conformation in membrane-mimetic environments. Isotope labeling of CRAMP is expected to make it possible to study the structure and dynamic properties of CRAMP in various membrane systems.

• Fisheries and Aquatic Sciences
• /
• 제25권7호
• /
• pp.357-379
• /
• 2022

Increased fisheries products have raised by-products that are discarded due to low economic value. In addition, marine by-products are still rich in protein and nutritional value that have biological activities and give benefits to human health. Meanwhile, there is raised pressure for sustainability practices in marine industries to reduce waste and minimize the detrimental effect on the environment. Thus, valorization by-products through bioactive peptide mining are crucial. This review focus on various ways to obtain bioactive peptides from marine by-products through protein hydrolysis, for instance chemical hydrolysis (acid and based), biochemical hydrolysis (autolysis and enzymatic hydrolysis), microbial fermentation, and subcritical water hydrolysis. Nevertheless, these processes have benefits and drawbacks which need to be considered. This review also addresses various biological activities that are favorable in pharmaceutical industries, including antioxidant, antihypertensive, anticancer, anti-obesity, and other beneficial bioactivities. In addition, some potential marine resources of Indonesia for the marine biopeptide from their by-product or undesired marine commodities would be addressed as well.

• 대한한의학방제학회지
• /
• 제26권3호
• /
• pp.251-259
• /
• 2018

Objective : Kurarinone is one of the flavonoids isolated from Sophorae Radix with various biological activities including anti-microbial effect. In this study, we investigated the effects of Kurarinone on tert-butyl hydroperoxide (tBHP)-induced oxidative stress finally leading to apoptosis in human hepatoma cell line HepG2. Methods : To determine the effects on cell viability, the cells were exposed to tBHP ($100{\mu}mol/l$) after pretreatment with kurarinone (0.5 and $1{\mu}g/ml$). Cell viability was measured by MTT assay. To reveal the possible mechanism of cytoprotectivity of kurarinone, levels of reactive oxygen species, intracellular glutathione, mitochondrial membrane potential, and expression of caspase were examined. Results : tBHP-induced cell death was due to oxidative stress and the resulting apoptosis. Kurarinone dose-dependently protected cells from apoptosis when determined by MTT and TUNEL assay. Consistent with this observation, decreased expression of pro-caspase 3/9 protein by tBHP was restored by kurarinone. Kurarinone also showed anti-oxidative effects by inhibiting generation of ROS and depletion of GSH in tBHP-stimulated HepG2 cells. In addition, kurarinone significantly recovered disruption of mitochondrial membrane potential (MMP) as a start sign of hepatic apoptosis induced by oxidative stress. Conclusion : From these results, it was concluded that kurarinone protected tBHP-induced hepatotoxicity with anti-oxidative and anti-apoptotic activities. Our results suggest that kurarinone might be beneficial to hepatic disorders caused by oxidative stress.

• IMMUNE NETWORK
• /
• 제6권2호
• /
• pp.102-110
• /
• 2006

Background: Dendritic cells (DC) are professional antigen-presenting cells in the immune system and can induce T cell response against virus infections, microbial pathogens, and tumors. Therefore, immunization using DC loaded with tumor-associated antigens (TAAs) is a powerful method of inducing anti-tumor immunity. For induction of effective anti-tumor immunity, antigens should be efficiently introduced into DC and presented on MHC class I molecules at high levels to activate antigen-specific $CD8^+$ T cells. We have been exploring methods for loading exogenous antigens into APC with high efficiency of Ag presentation. In this study, we tested the effect of the cationic liposome (Lipofectin) for transferring and loading exogenous model antigen (OVA protein) into BM-DC. Methods: Bone marrow-derived DC (EM-DC) were incubated with OVA-Lipofectin complexes and then co-cultured with B3Z cells. B3Z activation, which is expressed as the amount of ${\beta}$-galactosidase induced by TCR stimulation, was determined by an enzymatic assay using ${\beta}$-gal assay system. C57BL/6 mice were immunized with OVA-pulsed DC to monitor the in vivo vaccination effect. After vaccination, mice were inoculated with EG7-OVA tumor cells. Results: BM-DC pulsed with OVA-Lipofectin complexes showed more efficient presentation of OVA-peptide on MHC class I molecules than soluble OVA-pulsed DC. OVA-Lipofectin complexes-pulsed DC pretreated with an inhibitor of MHC class I-mediated antigen presentation, brefeldin A, showed reduced ability in presenting OVA peptide on their surface MHC class I molecules. Finally, immunization of OVA-Lipofectin complexes-pulsed DC protected mice against subsequent tumor challenge. Conclusion: Our data provide evidence that antigen-loading into DC using Lipofectin can promote MHC class I- restricted antigen presentation. Therefore, antigen-loading into DC using Lipofectin can be one of several useful tools for achieving efficient induction of antigen-specific immunity in DC-based immunotherapy.

• Journal of Periodontal and Implant Science
• /
• 제34권2호
• /
• pp.345-356
• /
• 2004

The triclosan was shown to have anti-microbial and anti-inflammatory effect with inhibition of inflammatory mediators such as prostaglandin $E_2(PGE_2)$. The purpose of this study was to elucidate whether and how $PGE_2$ could be inhibited by triclosan in human gingival fibroblast. Human gingival fibroblast-1 cells (ATCC CRL2014) were pre-treated for 1 hour with triclosan (0.001 ${\mu}/ml{\sim}10$ ${\mu}/ml$) and then stimulated with $TNF-{\alpha}$ (1.0 ng/ml). $PGE_2$ synthesis was evaluated by ELISA and gene expression of COX-1 and COX-2 was evaluated by RT-PCR after $TNF-{\alpha}$, triclosan, and NS-398 (COX-2 inhibitor, 5, ${\mu}M$) and/ or cycloheximide (protein synthesis inhibitor, 2 ${\mu}g/ml$). Triclosan was cytotoxic to human gingival fibroblasts in the concentration higher than 1.0 ${\mu}g/ml$ for longer than 24 hours in tissue culture. The $PGE_2$ synthesis was inhibited by triclosan in dose-dependent manner. Greater COX-2 mRNA suppression was observed with triclosan (0.1 ${\mu}g/ml$) than with $TNF-{\alpha}$ alone, without change in COX-1 gene expression. Inhibitory effects of triclosan on $PGE_2$ synthesis disappeared in presence of cycloheximide. This study suggests that triclosan inhibit prostaglandin $E_2$ at the level of COX-2 gene regulation and require de novo protein synthesis.

• Journal of Animal Science and Technology
• /
• 제45권6호
• /
• pp.987-996
• /
• 2003

본 실험은 수준별 산삼 배양액에 의한 반추위 내 미생물 발효성상에 미치는 영향에 대하여 조사하기 위해 실시되었다. 산삼 배양액의 수준에 따른 반추위 내 발효성상에 미치는 영향은 WGM을 3% 첨가한 처리구가 대조구 및 다른 WGM 수준 첨가 처리구에 비하여 미생물 단백질 합성량이 가장 높게 나타났다. 산삼 배양액 내에 존재하는 saponin의 영향으로 배양 초기 NH3-N 농도의 수준이 WGM를 첨가한 처리구가 대조구에 비해 낮은 경향(P〈0.05)을 보였으나, 미생물단백질 합성량은 WGM 처리구에서 6시간 이후 급격히 증가(P〈0.05)하는 경향을 나타내었다. 따라서 용해도가 높거나 급여 초기 사료의 이용율이 저하되는 급여 체계에서 산삼 배양액을 첨가는 초기 반추위 미생물의 이용율을 조절할 수 있을 것으로 사료된다. 반추위 내 프로토조아의 수는 WGM 처리구에서 전반적으로 배양 9시간까지 낮아졌고, 3% 처리구에서 가장 낮았는데(P〈0.05), 프로토조아 제거효과에 의해 반추위 미생물합성량 증진시키는 효과를 나타내었다. NDF와 ADF 소화율은 대조구와 모든 처리구에서 배양시간 경과에 따라 높아졌고, NDF 소화율은 대조구와 WGM 3% 처리구에서 그리고 ADF 소화율은 처리구별 차이가 없었다. Total VFA 생성량은 처리구가 대조구에 비해 낮았고, 배양 12시간부터는 5% 처리구에서 차이가 없게 나타났다. 따라서 용해도가 높거나 급여 초기 사료의 이용률이 저하되는 급여 체계에서 산삼 배양액을 첨가는 초기 반추위 미생물의 사료 이용률 증진과 프로토조아 제거효과에 의해 반추위 미생물합성량 증진에 도움이 될 수 있을 것이다.

• 대한임상독성학회지
• /
• 제21권2호
• /
• pp.81-91
• /
• 2023

Purpose: Edible insect extracts have been used as an alternative source for medicinal supplements due to their significant antioxidative and anti-inflammatory activity. Recent studies have reported that anti-microbial peptides from insects have neuroprotective effects on dopamine toxins. The purpose of this study was to investigate the protective functions of mealworm (Tenebrio molitor) extract (MWE) on N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced cellular toxicity in SH-SY5Y neuroblastoma cells. Methods: Cellular toxicity induced by the MPTP toxin and the impact of MWE on cell survival were analyzed using MTT assays. DAPI staining was performed to observe apoptotic phenomena caused by MPTP. Changes in caspase-3 activity and protein expression were observed using enzyme activity assays and western blot assays, respectively. Results: MWE exerted significant antioxidant activity, which was measured by both DPPH and ABTS radical assays, with a dose-dependent relationship. Furthermore, MWE resulted in cellular proliferation in SHSY5Y cells in a dose-dependent manner. Furthermore, MWE pretreatment significantly inhibited MPTP-induced cytotoxicity, with a dose-dependent relationship. The morphological characteristics of apoptosis and increased reactive oxygen species induced by MPTP were also significantly reduced by MWE pretreatment. Conclusion: MWE treatment significantly attenuated MPTP-induced changes in the levels of proteins associated with apoptosis, such as caspase-3 and PARP. These findings suggest that MWE exerts neuroprotective effects on human neuroblastoma SH-SY5Y cells subject to MPTP-induced dopaminergic neurodegeneration.

• Fisheries and Aquatic Sciences
• /
• 제13권4호
• /
• pp.284-293
• /
• 2010

To reduce anti-nutritional factors in plant protein sources for fish meal replacement in fish feeds, cottonseed and soybean meal (CS) were fermented with Aspergillus oryzae. A feeding trial was conducted to verify the effects of fermented CS (FCS) with phytase supplementation on gossypol detoxification, phosphorus digestibility, antioxidant activity, and growth performance of juvenile olive flounder over 10 weeks. Four diets were formulated to replace 0, 30, or 40% fish meal protein with CS or FCS (designated as CS0, CS30, FCS30P, and FCS40P). Phytase (1,000 FTU/kg) was added to FCS30P and FCS40P. The microbial fermentation significantly increased dietary total polyphenols and consequently led to higher DPPH radical-scavenging activities in fish feed and fish tissue. Dietary and liver gossypol concentrations were dramatically decreased by the fermentation process. Phosphorus digestibility was significantly increased in fish fed the FCS40P diet. However, growth performance decreased in fish fed FCS diets. This study demonstrates that the fermentation process and phytase supplementation can improve the phosphorus availability of plant protein sources in fish. The fermentation of CS by A. oryzae could increase antioxidant activities in feed and fish and effectively degrade toxic gossypol in cottonseed meal.