• Journal of Nutrition and Health
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• v.28 no.10
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• pp.1022-1030
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• 1995

Saponins are glycosidic compounds present in many plant foods. They are characterized by their ability to lyse cell membranes due to their surface-active properties. Saponins are believed to interact primarily with cholesterol in the cell membrane. In this study, the interaction of soybean(SS) with cell membrane was investigated using erythrocytes as a model. Mechanisms of interaction was also investigated by measuring their binding capacity with different membrane lipid fractions. Throughout the study, gypsophilla saponin(GS) and quillaja saponin(QS) were used to evaluate the membranolytic activity of soybean saponins. All saponins released hemoglobin in a concentration-dependent manner. SS induced 40% hemolysis at the concentration of 400 ppm, however there was no increase in hemoglobin release above 400ppm concentration. 5ppm of GS and 8 ppm of QS hemolyzed 100% of erythrocytes. Isolation of SS fractions by thin layer chromatography revealed that only one non-polar saponin possesses strong hemolytic activity. When saponins were incubated decreased the release of cholesterol. When the hemolytic activity of saponins was measured in the presence of other major membrane lipid components, sphingomyelin significantly reduced the hemolytic activity of SS, while cholesterol reduced the activity of QS. GS showed high affinity to other component(s) in the incubation media as well as lipids. These results suggest that the membranolytic activity of saponins are related to their specific chemical structure, which determines the interaction behavior between saponins and different membrane components, and thereby influence the biological activity.

• Journal of Life Science
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• v.28 no.1
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• pp.99-104
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• 2018

Streptococcus pneumoniae is one of the major pathogens in community-acquired diseases, and it contains several factors that promote its pathogenesis, including pneumolysin (PLY). PLY is a member of the cholesterol-dependent cytolysin family, which attacks cholesterol-containing membranes, thereby forming ring-shaped pores. Thus, it is a major key target for vaccines against pneumococcal disease. We cloned the PLY gene from S. pneumoniae D39 and inserted it into the pQE-30 vector. Recombinant PLY (rPLY) was overexpressed in Escherichia coli M15 and purified by $Ni^{2+}$ affinity chromatography. Similarly, a PLY-EGFP fusion gene was produced by inserting the EGFP gene at the 3' end of the PLY gene in the same vector, and the recombinant protein was purified. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) showed that both recombinant proteins were purified. rPLY exhibited significant hemolytic activity against 1% human red blood cells (RBCs). Complete hemolysis was obtained at 500 ng/ml, and 50% hemolysis was found with a 240 ng/ml concentration. In contrast, rPLY-EGFP did not show hemolytic activity. However, rPLY-EGFP did bind the RBC membrane, indicating that rPLY-EGFP lost hemolytic activity via EGFP fusion, while retaining its membrane-binding ability. These data suggest that PLY's C terminus is important for its hemolytic activity. Therefore, these two recombinant proteins can be extremely useful for investigating the toxin mechanism of PLY and cell damage during pneumonia.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.49 no.2
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• pp.137-145
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• 2016

This study screened the biological activity of an acidified gill extract of the Manila clam Ruditapes philippinarum including antimicrobial, hemolytic, membrane permeabilization, and DNA-binding activity, and purified the antimicrobial material. The acidified gill extract showed potent antimicrobial activity against Bacillus subtilis and Escherichia coli without significant hemolytic activity, but showed no membrane permeabilization or DNA-binding ability. An antimicrobial material was purified from the acidified gill extract using C₁₈ reversed-phase and cation-exchange high-performance liquid chromatography (HPLC). Treatment of the purified material with trypsin completely abolished all of the antibacterial activity against Bacillus subtilis, suggesting that the purified material is a proteinaceous antibiotic. The molecular weight of the purified material was 2571.9 Da, but no primary structural information was obtained due to N-terminal blocking. A future study should confirm the primary structure. Our results suggest that the Manila clam gill contains proteinaceous antibiotics that have a role in first-line defense. This information could be used to better understand the Manila clam innate immune system.

• Journal of Applied Biological Chemistry
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• v.61 no.4
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• pp.405-410
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• 2018

Pseudomonas tolaasii strain No. 6264 has been isolated from mushroom tissue and identified as one of the major pathogen causing brown blotch disease. It secretes peptide toxins, known as tolaasin and its analogue peptides. P. tolaasii 6264 has been used as a typical pathogenic strain to study the brown blotch disease for last 20 years after confirming its blotch-forming ability, hemolytic activity, and white line formation. In this study, the characteristics of P. tolaasii 6264 strain were analyzed and compared according to storage period. Strains of P. tolaasii 6264 stored annually since 2012 were cultured and their pathogenic characters were analyzed. When the 16S rRNA sequences were compared, all strains were divided into two groups. Pathogenic characters including hemolytic activity, blotch-forming ability, and white line test were also investigated. The strains, P. tolaasii 6264-15-2 and P. tolaasii 6264-17, had all three activities; however, the rest of stored strains showed only blotch-forming ability losing other pathogenic characters. Tolaasin peptides were purified from the bacterial cultures and analyzed by mass spectrometry. The strains, P. tolaasii 6264-15-2 and P. tolaasii 6264-17, secreted Tol I (1987 Da), Tol II (1943 Da), and its analogues (1973 Da, 2005 Da) while some of these peptides were not found in the media cultured other strains. These results indicate that the pathogenicity of P. tolaasii could be varied during the storage period.

• Journal of Pharmaceutical Investigation
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• v.23 no.2
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• pp.111-118
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• 1993

The hemolytic characteristics of amphotericin B-containing liposomes have been investigated in vitro. From the hemolysis of human erythrocytes against free and liposomal amphotericin B, the marked reduction in the toxicity of amphotericin B was observed by incorporating the drug in egg PC liposomes. For 45 min, free amphotericin B at $9.6\;{\mu}g/ml$ could completely lyse 2 wt% human erythrocytes. However, liposomal amphotericin B had essentially no lytic effect even in the range over $9.6\;{\mu}g/ml$. In the 66 hr-hemolysis experiment, liposomal amphotericin B showed the slowly hemolysing chracteristics during the experimental period regardless of the concentration of amphotericin B but rapid hemolysis only for 12 hr was observed in the case of free amphotericin B and the degree of hemolysis for 12 hr was maintained after that time. Also the hemolysing ability of liposomal amphotericin B at $4\;{\mu}g/ml$ was lower than that of free amphotericin B at the same concentration for 66 hr. On the other hand, the dependence of hemolysis on amphotericin B contents in egg PC liposomes was significant between 1.64 mole% amphotericin B-containing liposomes and 15.79 or 27.27 mole% amphotericin B-containing liposomes. But no marked difference in hemolysis was observed between 15.79 and 27.27 mole% amphotericin B-containing liposomes. Especially, cholesterol as an excipient in amphotericin B-containing liposomes significantly reduced the hemolysis of human erythrocyte. The degree of hemolysis in 5 mole% amphotericin B-containing liposomes was reduced to approximately 50% of value in the cholesterol-free liposomes by adding 50% cholesterol.

• Proceedings of the Korean Society of Tribologists and Lubrication Engineers Conference
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• 2002.10b
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• pp.371-372
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• 2002

It is well known that red blood cells (RBCs) are suffered from chronic stresses in systemic circulation. The objective of this study is to clarify the effect of the aging of RBCs on rheological properties and hemolysis. Initially, RBCs age fractionation was performed by using a high-speed centrifugation (15[min] at 1500[G]), then young and aged RBCs were suspended in plasma to adjust the hematocrit level of 40[%]. After this pretreatment, the viscosity was measured by using a capillary type and a cone-plate type viscometers, respectively, and the hemolysis test was carried out by a seesaw type shaker. Results from these experiments showed that the viscosity of the aged RBCs measured by the capillary viscometer was increased by 10[%] as compared with that of the young RBCs. Under the condition of all shear zones, the viscosity of the aged RBCs was increased in case of using the cone-plate type viscometer. And the hemolytic level was increased twice as the aging. The data obtained in this study indicated that the ability of aggregation of RBCs was increased and the deformability of RBCs membrane got lower with the aging. Furthermore, it was exhibited that the fragility of RBCs ’ membrane was increased with the aging.

• Journal of Microbiology and Biotechnology
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• v.30 no.3
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• pp.382-390
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• 2020

Periplanetasin-4 is an antimicrobial peptide with 13 amino acids identified in cockroaches. It has been reported to induce fungal cell death by apoptosis and membrane-targeted action. Analogs were designed by substituting arginine residues to modify the electrostatic and hydrophobic interactions accordingly and explore the effect of periplanetasin-4 through the increase of net charge and the decrease of hydrophobicity. The analogs showed lower activity than periplanetasin-4 against gram-positive and gram-negative bacteria. Similar to periplanetasin-4, the analogs exhibited slight hemolytic activity against human erythrocytes. Membrane studies, including determination of changes in membrane potential and permeability, and fluidity assays, revealed that the analogs disrupt less membrane integrity compared to periplanetasin-4. Likewise, when the analogs were treated to the artificial membrane model, the passage of molecules bigger than FD4 was difficult. In conclusion, arginine substitution could not maintain the membrane disruption ability of periplanetasin-4. The results indicated that the attenuation of hydrophobic interactions with the plasma membrane caused a reduction in the accumulation of the analogs on the membrane before the formation of electrostatic interactions. Our findings will assist in the further development of antimicrobial peptides for clinical use.

• Korean Journal of Veterinary Research
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• v.30 no.4
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• pp.427-434
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• 1990

The purpose of this study was to examine O serotypes, colicin and hemolysin production. antibiotic susceptibility and haemagglutinating ability to animal erythrocytes among Escherichia coli strains isolated from pigeons in Taegu province. Of the 166 strains isolated, 28 strains (16.9%) were classified into 6 O serotypes and their types were O20(42.9%), O15(17.9%), O139(14.3%), O101(10.7%), O149(7.1%) and O8(7.1%). Of the 166 strains isolated, none was hemolytic and 3(1.8%) were colicinogenic. Antibiotic susceptibility test of Escherichia coli isolates was performed by the agar dilution method, using ampicillin, chloramphnicol, gentamicin, rifampicin, streptomycin (Sm), nalidixic acid, sulfadimethoxine and tetracycline (Tc). Forty four strains (26.5%) were resistant to one or more drugs and the most common resistance patterns were SmTc (27.3%). Of the 44 drug resistant strains, 6 strains haemagglutinated erythrocytes of chicken, guinea pig and 2 of the 6 strains agglutinated goose erythrocytes.

• Microbiology and Biotechnology Letters
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• v.38 no.1
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• pp.19-23
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• 2010

Recently, marine organisms are emerging as a leading group for identifying and extracting novel bioactive substances. These substances are known to possess a potential regarding not only as a source of pharmaceutical products but also their beneficial effects on humans. Among the substances, antimicrobial peptides (AMPs) specifically have attracted considerable interest for possible use in the development of new antibiotics. AMPs are characterized by relatively short cationic peptides containing the ability to adopt a structure in which cationic or hydrophobic amino acids are spatially scattered. Although a few reports address novel marine organisms-derived AMPs, their antimicrobial mechanism(s) are still remain unknown. In this review, we summarized the peptides previously investigated, such as Pleurocidin, Urechistachykinins, Piscidins and Arenicin-1. These peptides exhibited significant antimicrobial activities against human microbial pathogens without remarkable hemolytic effects against human erythrocytes, and their mode of actions are based on permeabilization of the plasma membrane of the pathogen. Therefore, the study of antimicrobial peptides derived from marine organisms may prove to be useful in the design of future therapeutic antimicrobial drugs.

• Mycobiology
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• v.36 no.1
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• pp.50-54
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• 2008

This study examined the chemical composition of A. blasiliensis and the chemical structural properties of an immuno-stimulating polysaccharide. The amino acids, free sugars, and organic acids by HPLC and fatty acids by GC were analyzed. The immuno-stimulating substance from A. blasiliensis was extracted with hot water and purified by ethanol precipitation. It underwent ion exchange chromatography on DEAE-cellulose and gel filtration on Toyopearl HW 65F. Through GP-HPLC, the substance was found to be homogeneous. Its chemical structure was determined by $^{13}C-NMR$. Fatty acids, organic acids, and sugar alcohol composition consisted exclusively of linoleic acid, fumaric acid and mannitol, respectively. The amino acids were mainly glutamic acid, glycine, and arginine. By $^{13}C-NMR$ analysis, the immuno-stimulating substance was identified as ${\beta}-(1{\rightarrow}3)\;(1{\rightarrow}6)$-glucan, composed of a backbone with $(1{\rightarrow}3)$-linked D-glucopyranosyl residues branching a $(1{\rightarrow}6)$-linked D-glucopyranosyl residue. The ${\beta}$-glucan from A. blasiliensis showed pronounced immuno-stimulating activity on the antibody-production ability of B-lymphocytes by the hemolytic suspension assay. In these results, A. blasiliensis was estimated to have potent pharmacological properties and potential nutritional values.