• Fisheries and Aquatic Sciences
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• 제26권3호
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• pp.204-215
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• 2023

The thermolabile haemolysin (tlh) of Vibrio parahaemolyticus (Vptlh) from V. parahaemolyticus is a multiple-function enzyme, initially describes as a haemolytic factor activated by lecithin and phospholipase A2 enzymatic activity (Shinoda, 1991; Vazquez-Morado, 2021; Yanagase et al., 1970). Until now, the tlh structure has hypothesized including N-terminal and C-terminal domain, but what domain of the Vptlh structure does the haemolytic activity has not been refined yet. In this study, a 450-bp VpTLH nucleotide sequence of the entire Vptlh gene encoded the C-terminal domain cloned firstly to examine its responsibility in the activity of the Vptlh. The C-terminal domain fused with a 6-His-tag named the His-tag-VpC-terminal domain was expressed successfully in soluble form in the BL21 (DE3) PlysS cell. Remarkably, both expression and purification results confirmed a high agreement in the molecular weight of the His-tag-VpC-terminal domain was 47 kDa. This work showed the His-tag-VpC-terminal domain lysed the erythrocyte membranes in the blood agar and the phosphate buffered saline (0.9%) media without adding the lecithin substrate of the phospholipase enzyme. Haemolysis occurred at all tested diluted concentrations of His-tag-VpC-terminal domain (p < 0.05), providing evidence for the independent haemolytic activity of the His-tag-VpC-terminal domain. The content of 100 ㎍ of the His-tag-VpC-terminal domain brought the highest haemolytic activity of 80% compared to that in the three remaining contents. Significantly, the His-tag-VpC-terminal domain demonstrated not to involve the phospholipase activity in Luria-Bertani agar supplemented with 1% (vol/vol) egg yolk emulsion. All results proved the vital responsibility of the His-tag-VpC-terminal domain in causing the haemolytic activity without the required activation by the phospholipase enzyme. Raw extracts of Phellinus igniarus and Phellinus pipi at 10^-1 mg/mL inhibited the haemolytic activity of the His-tag-VpC-terminal domain from 67.7% to 87.42%, respectively. Hence applying the His-tag-VpC-terminal domain as a simple biological material to evaluate quickly potential derivatives against the Vptlh in vivo conditions will accessible and more advantageous than using the whole of the Vptlh.

• 한국환경성돌연변이발암원학회지
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• 제26권2호
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• pp.48-52
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• 2006

Our investigation of the seaweed extracts, Ulva lactuca. The biological activities antioxidant, antimicrobial activity, antifungal and haemolytic activity of ethy-ether and ethyl-acetate extracts from the seaweed, Ulva lactuca were investigated. They were separately extracted using ethyl-ether and ethyl-acetate from dried samples at room temperature and freeze dried. Seaweed extracts were found to cause significant free radical scavenging effects on DPPH (1,1-diphenyl-2-picrylhydrazyl). Seaweed extracts had not significant haemolytic activity against human erythrocyte. This extracts exhibited in vitro broad-spectrum antimicrobial activity of gram-negative, gram-positive bacteria and without antifungal activity.

• IMMUNE NETWORK
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• 제8권3호
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• pp.67-74
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• 2008

Background: The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the rainbow trout were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of rainbow trout, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. Methods: The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at $10^8$ cfu/g Pdp11, $10^8$ cfu/g 51M6 or with $0.5{\times}10^8$ cfu/g Pdp11 plus $0.5{\times}10^8$ cfu/g 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, and then the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. Results: The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in rainbow trout fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. Conclusion: Our results demonstrated the usefulness of incorporating inactivated probiotic bacteria into fish diets.

• 한국어병학회지
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• 제15권2호
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• pp.77-82
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• 2002

Effects of stress-induced suppression of humoral immunity on scuticociliate killing activity of olive flounder plasma were investigated. Changes in glucose level. alternative complement activity and Iysozyme activity of plasma by handling stress were analysed in relation to in vitri parasiticidal activity of plasma. The plasma glucose level was about two times higher in fish after a handling stress than in control fish. Plasma lysozyme activity and natural haemolytic activity were decreased in suessed fish.The scuticociliate killing activity of plasma was significantly lower in stressed fish than in non-stressed control fish. The present results indicated that stress-induced immunodepression could be a cause of scuticociliatosis occurrence in olive flounder.

• 한국미생물·생명공학회지
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• 제50권1호
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• pp.51-62
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• 2022

Lactobacillus rhamnosus SKG34 (LrSKG34), a potential probiotic strain, was successfully isolated from Sumbawa Mare's milk. Our previous studies showed that the strain is resistant to gastrointestinal conditions, possesses antioxidant activity, and lowers blood cholesterol levels. Further clarification of the potential probiotic characteristics and safety assessment are necessary. This study aimed to evaluate the adhesion of LrSKG34 to Caco-2 cell monolayers and its effect on mucosal integrity in vitro. We also examined the LrSKG34 safety profile based on antimicrobial susceptibility testing, haemolytic activity determination, Caco-2 cell monolayer translocation evaluation, and in vivo investigation of the effect of LrSKG34 on the physiology, biochemical markers, and histopathological appearance of major organs in an animal model. LrSKG34 attached to Caco-2 cell monolayers and maintained mucosal integrity in vitro. The typical resistance of lactobacilli to ciprofloxacin, gentamicin, vancomycin, trimethoprim-sulfamethoxazole, and metronidazole was confirmed for LrSKG34. No haemolytic activity was observed on blood agar plates, and no LrSKG34 translocation was observed in Caco-2 cell monolayers. Administration of LrSKG34 to Sprague-Dawley rats did not adversely affect body weight. No abnormalities in hematological parameters, serum biochemistry levels, or histopathological structures of major organs were observed in LrSKG34-treated rats. Collectively, the results implicate LrSKG34 as a promising and potentially safe probiotic candidate for further development.

• 약학회지
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• 제42권3호
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• pp.248-256
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• 1998

We had already reported that we purified N-${\beta}$-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), a novel antibacterial substance from the immunized adult Sarcoph aga peregrina (Flesh fly). We found that the antibacterial activity of synthetic 5-S-GAD is equal to that of authentic 5-S-GAD without a specificity of antibacterial activity against Gram positive and Gram negative. Significant synergism was detected between 5-S-GAD and streptomycin against streptomycin resistant strain E.coli K12 594. It has an antitumor activity against several tumor cell lines at a concentration of $100{\mu}M$. However, no cytotoxic activity against murine macrophage was detected at a concentration of $500{\mu}M$. Furthermore, haemolytic activity against sheep erythrocytes was not detected at the same concentration. We suggest that the S-conjugation of glutathion with dihydroxyphenylalanine might be important to increase antibacterial activity of dihydroxyphenylalanme.

• 한국환경보건학회지
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• 제8권1호
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• pp.81-97
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• 1982

Ethylene oxide gas has been used as a cold sterilant for heat-sensitive medical equipments and as a fumigant for food for more than 30 years, and it is used more widely than radiation although radiation sterilization has made significant inroads in recent years. But according to recent studies of toxicities such as mutagenicity, haemolytic effect and possible carcinogenicity of Ethylene oxide (ETO) and its two main reaction products, Ethylene chlorohydrin (ETCH) and Ethylene glycol (ETG), Environmental Protection Agency in U.S.A. has suggested some regulations on residual gas in drug products and medical devices for human use. The mutagenic activity of ETO compared with that of X-ray has an equivalency of 1 ppm/hr for ETO as compared to 20 mrad for X-ray, and one could suggest the present maximum allowable concentration for ETO (50 ppm) should be 400 times lower than the radiation standard (2.5 mrad/hr). Although radiation sterilization has advantages of simplicity of operation and complete reliability, changes of physico-chemical properties with possible formation of toxic substances may occur. It is therefore necessary to make some regulations of our own for residual toxicities orginated from each sterilization method.

• 한국어병학회지
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• 제21권3호
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• pp.247-257
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• 2008

To understand the activity of non-specific defence factors in cultured Sebastes, the antibacterial effect of the serum, skin mucus and homogenate of various organs from cultured oblong rockfish (Sebastes oblongus) and rockfish(Sebastes schlegeli) against pathogenic bacteria, Aeromonas hydrophila, Edwardsiella tarda, Vibrio anguillarum, and Streptococcus sp. was compared with that of flounder(Paralichthys olivaceus) and seabass(Leteolabrax japonicus). And the activities of proteolytic enzyme, chitinolytic enzyme and haemolycin as non-specific defence factor were investigated on the oblong rockfish and rockfish. Samples from oblong rockfish showed the highest antibacterial activity by lysoplate assay on agar plate mixed with pathogens, followed in descending order by rockfish, seabass, and flounder. Turbidimetric assay was carried to evaluate the lysozyme activity of fish samples against lyophilized cells of Micrococcus lysodeiktikus. The serum, kidney, liver, stomach, intestine and eyeball of oblong rockfish and the mucus and gill of rockfish appeared to have the highest lysozyme activity among the fish strains investigated. All samples except skin mucus, liver, and eyeball of oblong rockfish and rockfish showed proteolytic enzyme activity. Chitinolytic enzyme activity was showed in random sampling and haemolytic activity was remarkable in oblong rockfish. Therefore, Sebastes strain was proved to have effective defense mechanisms based on the antibacterial activities, and lysozyme, proteolytic enzyme, chitinolytic enzyme, and haemolycin were considered to act as the non-specific defence factor of Sebastes.

• 한국어병학회지
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• 제20권2호
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• pp.99-108
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• 2007

본 연구는 V. harveyi의 luminescence 유무와 해수 어류에 대한 병원성의 관계를 알아보고자 non-luminous V. harveyi 와 luminous V. harveyi 분리 균주로 조피볼락에 대한 병원성 시험을 시행하였다. 시험 균주 V. harveyi 모두 haemolytic activity를 나타내었으며, 어체 내 생존능은 non-luminous V. harveyi 분리 균주 FR 2가 강한 생존능을 나타내었다. 또한 조피볼락에 인위 감염시, non-luminous V. harveyi 분리 균주 FR 2가 강한 병원성을 나타내었으며 luminous V. harveyi 분리 균주 VIB 391은 낮은 병원성을 나타내었다. 결론적으로 조피볼락에서는 non-luminous V. harveyi 분리 균주 FR 2가 강한 병원성을 나타내었으며 luminous V. harveyi 분리 균주 VIB 391은 매우 약한 병원성을 나타내어 luminescence의 유무에 따라 해수 어종에 대한 병원성의 차이가 있을 것으로 생각된다

• 한국수산과학회지
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• 제34권4호
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• pp.365-369
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• 2001

2000년도 2회에 걸쳐 제주도 일원 넙치 양식장을 대상으로 감염어에서 분리된 연쇄구균에 대한 특성시험 결과를 요약하면 다음과 같다. 감염어로부터 분리된 연쇄구균은 형태적으로 연쇄상의 gram 양성의 구균이었고, catalase와 oxidase에 음성이었으며, 용혈성 시험에서는 $\beta$-용혈성을 나타내었다. 감염어에서 각 장기별 생균수에서는 환부, 신장, 혈액 그리고 뇌순으로 연쇄구균이 검출되었다. 약제 감수성에서는 ampicillin, ciprofloxicin 둥에 감수성이 있었지만 erythromycin 등에는 감수성이 없었다. 복강에 주사하여 병원성 시험을 한 결과 10일 이내에 전량 폐사하는 강한 병원성을 보였다. 감염균의 용혈성에 대한 특성은 $50^{\circ}C$ 30분의 배양에서 용혈활성이 마르게 증가되어 최대값에 도달되었고 또한 $pH 5.5\~6.5$에서 높은 활성값을 보였다.