• The Korean Journal of Zoology
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• v.34 no.1
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• pp.20-30
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• 1991

지렁이(L. rubelIus) 체액내의 적혈구용혈능깍 단백질분해능을 확인하였다. 지렁이의 체액 0. 33 $\mu$l속에 있는 용혈인자는 9 $\times$ 1011 rat RBCs를 2분만에 완전히 용혈시켰으며, 다른 포유류들의 적혈구에도 약간의 차이는 있으나 비슷한 용혈능을 보였고, 이 용혈인자는 지렁이의 혈구가 아닌 몸체조직에서 분비되는 것으로 생각되었다. 용혈인자는 pH 6.5-7.5사이에서 활성이 가장 강하였고, 63'c로 30분 열처리할 경우 활성이 완전히 없어졌으며, 2-mercaptoethanol은 용혈인자의 활성을 증가시켰다. 이 인자의 활성도는 여러가지의 당류, LPS, cholesterol, prosphatidyl Choline, Ch10ropromazine, Sphin90myelin 및 FG2+, Fe3+, CU2+, ZR2+ 등의 금속이온에 의하여 활성이 저해되었다. 한편 지렁이 체액내의 단백질 분해인자는 BSA와 19G를 여러 조각으로 분해시켰으며 이 분해인자는 PMSF및 TLCK에 의하여 활성이 억제되지 않았다. 지렁이의 체강에는 용혈소를 분비하는 박테리아들이 존재하였으나 이들 박테리아들의 용혈소는 지렁이의 용혈인자와는 전기영동이동도에서 차이가 있었다.

• Restorative Dentistry and Endodontics
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• v.30 no.4
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• pp.335-343
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• 2005

Hemolytic property is a specific feature of bacteria to obtain iron which is essential for its survival in host tissues. Therefore, it is thought to be one of several factors of virulence. The purpose of this study was to investigate the hemolytic properties of Prevotella nigrescens isolated from the teeth diagnosed as pulp necrosis and apical periodontitis under the presence of hemolysin inhibitors such as $NaN_3$ and dithiothreitol. heat, various pH and cultural conditions. The results were as follows; 1. Clinically isolated P. nigrescens strains and standard P. nigrscens ATCC 33563 showed hemolytic activity. 2. P. nigrescens showed higher hemolytic activity against human erythrocytes than sheep or horse erythrocytes. 3. $NaN_3$ and dithiothreitol (DTT) reduced the hemolytic activity of P. nigrescens in a dose dependent manner (p<0.05). 4. Optimal pH for the maximum hemolytic activity of P. nigrescens was 4.0 and the hemolysin was stable under the $50^{\circ}C$, but the hemolytic activity was significantly decreased at $95^{\circ}C$. 5. P. nigrescens cultured in $10\%\;CO_2$ condition showed higher hemolytic activity than the bacteria cultured in the anaerobic condition.

• Applied Biological Chemistry
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• v.43 no.3
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• pp.190-195
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• 2000

Tolaasin is a peptide toxin produced by Pseudomonas tolaasii and causes a brown blotch disease forming brown, slightly sunken spots and blotches on the cultivated mushrooms. It is a lipodepsipeptide consisting of 18 amino acids and its molecular mass is 1,985 Da. It forms a pore in plasma membranes, resulting in the disruption of membranes of fungal, bacterial, plant, and animal cells as well as mushroom tissue. In order to measure the toxicity of tolaasin, erythrocytes of blood were used to evaluate the tolaasin-induced hemolysis. Hemolytic activity of tolaasin was measured by observing the absorbance change either at 420 nm, representing the release of hemoglobins from red blood cells(RBCs), or at 600 nm, representing the density of residual cells. The hemolytic activity of culture-extract of P. tolaasii increased at early-stationary phase of growth and was maximal at late stationary phase. The hemolytic activity of tolaasin appeared high in the RBCs of dog and rat. The RBCs of rabbit and hen were less susceptible to tolaasin. The effects of various cations were also measured. $Cd^{2+}$ and $La^{3+}$. as well as $Zn^{2+}$ appeared inhibitory to the tolaasin-induced hemolysis. The effects of various anions on tolaasin-induced hemolysis were measured and carbonate showed the greatest inhibition to the hemolysis. However, phosphate stimulated the tolaasin-induced hemolysis and no effects were observed by chloride and nitrate.

• Korean Journal of Microbiology
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• v.33 no.3
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• pp.170-174
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• 1997

In order to design a novel synthetic peptide with improved fungicidal activity but low hemolytic activity, a hybrid peptide, cecropin A(l-8)-magainin 2(1-12), and its analogue peptides were synthesized by the solid phase method. Antifungal and hemolytic activities of the synthetic peptides were assessed by the growth inhibition against Trichosporon beigelii and the cell membrane lysis against human red hlood cells, respectively. Analogue 2 in which Lys at position 12 in cecropin A(1-8)-magainin 2(1-12) was substituted with Ala showed most potent antifungal activity (MIC: 2.5.$\mu$g/ml) with minimal hemolytic activity (0.5% hemolysis at the (200.$\mu$g/ml peptide). This peptide (A2), therefore, could be useful as a model for further designing potent antifungal peptides without cytotoxicity.

• Journal of Applied Biological Chemistry
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• v.52 no.1
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• pp.28-32
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• 2009

The bacterial toxin, tolaasin, causes brown blotch disease on the cultivated mushrooms by collapsing fungal and fruiting body structure of mushroom. Cytotoxicity of tolaasin was evaluated by measuring hemolytic activity because tolaasins form membrane pores on the red blood cells and destroy cell structure. While we investigated the inhibitions of hemolytic activity of tolaasin by $Zn^{2+}$ and $Cd^{2+}$, we found that $Ni^{2+}$ is another antagonist to block the toxicity of tolaasin. $Ni^{2+}$ inhibited the tolaasin-induced hemolysis in a dose-dependent manner and its Ki value was $\sim10$ mM, implying that the inhibitory effect of $Ni^{2+}$ is stronger than that of $Cd^{2+}$. The hemolytic activity was completely inhibited by $Ni^{2+}$ at the concentration higher than 50 mM. The effect of $Ni^{2+}$ was reversible since it was removed by the addition of EDTA. When the tolaasin-induced hemolysis was suppressed by the addition of 20 mM $Ni^{2+}$, the subsequent addition of EDIA immediately initiated the hemolysis. Although the mechanism of $Ni^{2+}$ -induced inhibition on tolaasin toxicity is not known, $Ni^{2+}$ could inhibit any of fallowing processes of tolaasin action, membrane binding, molecular multimerization, pore formation, and massive ion transport through the membrane pore. Our results indicate that $Ni^{2+}$ inhibits the pore activity of tolaasin, the last step of the toxic process.

• The Korean Journal of Mycology
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• v.28 no.2
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• pp.123-125
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• 2000

To investigate the hemolytic toxicities of the Korean basidiomycetes, the cold-water extracts of 22 wild mushrooms were tested for their hemolytic activities on the erythrocytes of a mouse and a rat. As a result, three species including Collybia confluence, Laccaria vinaceoavellanea and Lenzites betulina showed strong hemolytic activities. Of these, L. vinaceoavellanea retained more than 80% of its hemolytic activity even after it was boiled for 30 minutes, while the other two were inactivited in five minutes.

• Applied Biological Chemistry
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• v.50 no.4
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• pp.257-261
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• 2007

Tolaasin, a peptide toxin produced by Pseudomonas tolaasii, causes a serious disease on the cultivated mushrooms, known as brown blotch disease. Hemolysis using red blood cells was designed to measure the cytotoxicity of tolaasin molecules. Since tolaasin has two amine groups near the C-terminus, its membrane binding will be dependent on the ionic states of the amine groups. When the tolaasin peptide was titrated, its titration curve indicated the presence of titratable amine(s) at pH ranges from 7.0 to 9.6. When the pH-dependence of tolaasin-induced hemolysis was measured at various pHs, hemolysis was more efficient at alkaline pHs. In order to measure the membrane binding activity of tolaasin at different pHs, RBCs were incubated with tolaasin molecules for short time periods and washed out with fresh buffer. Because of the tolaasin binding during the preincubation period, fast hemolyses were observed at pH 8 or higher. These results imply that non-charged or less positively charged states of tolaasin molecules easily bind to membrane and show high hemolytic activity.

• The Korean Journal of Mycology
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• v.25 no.4 s.83
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• pp.253-256
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• 1997

To elucidate the hemolytic toxicities of Korean basidiomycetes, the cold-water extracts of 46 wild mushrooms were tested for hemolytic activity on mouse erythrocytes. Twelve species including Amanita esculenta, A. griseofarinosa, A. longistriata, A. melleiceps, A. phalloides, A. rubescence, A. spissacea, A. vaginata, Hebeloma crustuliniforme, Lepiota cygnea, Macrolepiota procera, and Oudemansiella platyphylla showed strong hemolytic activities. Of these, A. griseofarinosa and H. crustuliniforme showed no decrease in their hemolytic activity even after five minutes' boiling.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.34 no.5
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• pp.502-508
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• 2001

The extracellualr hemolysin produced by pathogenic Vibrio furnissii was isolated and purified by ammonium sulfate precipitation and followed by DEAE-Sephadex A-50 (1st and 2nd) ion-exchange and Sephadex G-100 gel permeation chromatography. The molecular weight of the purified hemolysin was 61 kDa. The enzyme activity was inactivated by heating at 60 for 5 min, and inhibited by additions of $Cu^{2+},\;Zn^{2+}$ and a high concentration of cholesterol. Lysis of human erythrocytes by the enzyme was temperature dependent, and optimal temperature of the enzyme was $37^{\circ}C$. The purified hemolysin was unstable at pH 6.0, 6.5 and 10.0. The optimal concentration of human erythrocytes on hemolysins was about $1.5\%$. The purified hemolysin was active for erythrocytes from three animal species including mouse, rabbit and rat, and was the most active against rabbit erythrocytes. B blood group of human erythrocytes also showed a high sensitivity to the enzyme.

• Korean Journal of Microbiology
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• v.34 no.1_2
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• pp.26-30
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• 1998

Tetradecapeptide, Mastoparan B(MP B) and its [$Ala^2$]-, [$Ala^4$]-, [$Ala^6$]-, [$Ala^9$]-MP B derivatives were synthesized, and then their antibacterial and hemolytic activities were assayed to examine the effect of hydrophobicity and amphiphilicity on the MP B-induced those activities. MP B and more hydrophobic [$Ala^2$]-, [$Ala^4$]-MP B showed stronger antibacterial activity and less hydrophobic [$Ala^6$]-MP B than MP B did similar or weaker activity, so more hydrophobic [Ala]-MP B derivative had stronger activity. But more hydrophobic [$Ala^9$]-MP B than MP B showed weaker activity because of its Trp substitution by Ala. On the other hand, [$Ala^2$]- and [$Ala^4$]-MP B showed 100.0% and 69.4% hemolytic activity, but [$Ala^6$]-MP B did the weakest activity(6.1%) and [$Ala^9$]-MP B, weaker activity(26.0%) than MP-B. Therefore, more hydrophobic [Ala]-MP B derivative had stronger activity and the effect of amphiphilicity on the activity was weak.