• YAKHAK HOEJI
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• v.43 no.6
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• pp.802-808
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• 1999

Vibrio vulnificus cytolysin has been incriminated as one of the important virulence determinants in V. vulnificus infection. In the present study, the effects of Vibrio vulnificus cytolysin on platelets were examined. Vibrio vulnificus cytolysin induced platelet aggregation and increased intracellular calcium concentration ($[Ca^{2+}]_i$) of rat platelets. These effects were abolished in $Ca^{2+}-free$ buffer (2 mM EGTA). Cytolysin also potentiated ADP-and collagen-induced platelet aggregation. Lanthanum (2 mM) inhibited cytolysin-diduced platelet aggregation. However, another $Ca^{2+}$ channel blockers, verapamil ($20{\;}{\mu}M$) or mefenamic acid ($20{\;}{\mu}M$) did not block cytolysin-induced platelet aggregation. Osmotic protectants, sucrose (50 mM) and raffinose (50 nM) suppressed platelet aggregation by 35.9% and 63.4%, respectively. V. vulnificus cytolysin increased membrane conductances of platelet membranes. These results suggest that cytolysin-induced platelet aggregation is mediated via lanthanum sensitive-calcium influx which resulted from the pore formation by V. vulnificus cytolysin.

• BMB Reports
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• v.32 no.3
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• pp.273-278
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• 1999

Cytolysin produced by Vibrio vulnificus has been known to be lethal to mice by increasing vascular permeability and neutrophil sequestration in the lung. In the present study, a cytotoxic mechanism of V. vulnificus cytolysin on the neutrophil was investigated. Cytolysin rapidly bound to neutrophils and induced cell death, as determined by the trypan blue exclusion test. V. vulnificus cytolysin caused the depletion of cellular ATP without the release of ATP or lactate dehydrogenase. Formation of transmembrane pores was evidenced by the rapid efflux of potassium and 2-deoxy-D-[$^3H$]glucose from cytolysin-treated neutrophils. It was further confirmed by the rapid flow of monovalent ions in the patch clamp of cytolysin-treated neutrophil membrane. The pore formation was accompanied by the oligomerization of cytolysin monomers on the neutrophil membrane as demonstrated by immunoblot, which exhibited a 210 kDa band corresponding to a tetramer of the native cytolysin of $M_r$ 51,000. These findings indicate that V. vulnificus cytolysin rapidly binds to the neutrophil membrane and oligomerizes to form small transmembrane pores, which induce the efflux of potassium and the depletion of cellular ATP leading to cell death without cytolysis.

• Toxicological Research
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• v.4 no.2
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• pp.143-149
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• 1988

Cytolysin produced by Vibrio vulnificus ATCC 27562 was partially purified by sequential ammonium sulfate precipitation, gel filtration with Sephadex G-200, and ion exchange chromatography with DEAE-Sephadex. The partially purified cytolysin was inactivated by cholesterol. More than one molecule of the cytolysin was required to lyse a single erythrocyts. The antiserum against cytolysin enhanced the survival ratio of mice infected with low dose of V. vulnificus.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.5
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• pp.259-264
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• 2004

Cytolysin produced by Vibrio vulnificus has been incriminated as one of the important virulence determinants in V. vulnificus infection. Ion selectivity of cytolysin-induced pores was examined in a CPAE cell, a cell line of pulmonary endothelial cell, using inside-out patch clamp techniques. In symmetrical NaCl concentration (140 mM), intracellular or extracellular application of cytolysin formed ion-permeable pores with a single channel conductance of $37.5{\pm}4.0$ pS. The pore currents were consistently maintained after washout of cytolysin. Replacement of $Na^+$ in bath solution with monovalent ions $(K^+,\;Cs^+\;or\;TEA^+)$ or with divalent ions $(Mg^{2+},\;Ca^{2+})$ did not affect the pore currents. When the NaCl concentration in bath solution was lowered from 140 to 60 and 20 mM, the reversal potential shifted from 0 to -11.8 and -28.2 mV, respectively. The relative permeability of the cytolysin pores to anions measured at $-40\;mV\;was\;Cl^-\;=\;NO_2^-\;{\geq}\;Br^-\;=\;I^-\;> \;SCN^-\;>\;acetate^-\;>\;isethionate^-\;>\;ascorbic acid^-\;>\;EDTA^{2-},$ in descending order. The cytolysin-induced pore current was blocked by $CI^-$ channel blockers or nucleotides. These results indicate that V. vulnificus cytolysin forms anion-selective pores in CPAE cells.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.2
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• pp.199-205
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• 1999

Vibrio vulnificus cytolysin caused platelet cytolysis and increased intracellular calcium concentration $([Ca^{2+}]_i)$ of rat platelets in a concentration-dependent manner. In the presence of V. vulnificus cytolysin (3 HU/ml), lactate dehydrogenase (LDH) activity was increased from $1.3{\pm}0.4%$ of control to $64.3{\pm}3.4%$ in platelet suspension buffer. In $Ca^{2+}-free$ platelet suspension buffer, however, V. vulnificus cytolysin did not induce $[Ca^{2+}]_i$ increase and LDH release. Addition of EGTA (2 mM) to suspension buffer after the initial $Ca^{2+}$ influx reversed $[Ca^{2+}]_i$ to the control level. However, a $Ca^{2+}$ channel blocker verapamil $(20\;{\mu}M)$ or mefenamic acid $(20\;{\mu}M)$ did not inhibit V. vulnificus cytolysin-induced $[Ca^{2+}]_i$ increase and LDH release. Divalent cations such as $Co^{2+},\;Cd^{2+}\;or\;Mn^{2+}$ (2 mM each) also did not alter V. vulnificus cytolysin-induced $[Ca^{2+}]_i$ increase and LDH release. V. vulnificus cytolysin (3 HU/ml)-induced calcium influx was completely blocked by lanthanum (2 mM). Lanthanum (2 mM) also completely blocked V. vulnificus cytolysin (3 HU/ml)-induced LDH release. Osmotic protectants such as, raffinose, sucrose or PEG600 (50 mM each) did not inhibit the lytic activity of V. vulnificus cytolysin. In conclusion, lanthanum sensitive $Ca^{2+}$ influx plays a significant role in Vibrio vulnificus cytolysin-induced platelet cytolysis and thrombocytopenia in V. vulnificus infection.

• Proceedings of the Korean Biophysical Society Conference
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• 1997.07a
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• pp.34-34
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• 1997

Vibrio vulnificus is an estuarine bacterium that has been associated with septicemia and serious wound infection in person. Cytolysin has been incriminated as one of the important virulence determinants. Little is known about the target cell of Vibrio vulnificus cytolysin in the body. Recently, we observed cytolysin-induced blood coagulation in rat.(omitted)

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.281-281
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• 1996

The immunoassay method is frequently used for the identification and quantitation of ultratrace amount of bioactive substances. Homogeneous liposome immunoassays, which can avoid the use of radioisotopes and separation steps, have recently been reported in many publications. Cytolysin-mediated liposome immunoassay using melittin ever been studied but showed limited applications. Here, we designed a homogeneous liposome immunoassay using Clostridium perfringens phospholipase C (PLC), an enzyme which catalyzes the hydrolysis of phosphatidylcholine in biological membranes, as a cytolysin.

• Journal of Food Hygiene and Safety
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• v.21 no.4
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• pp.213-217
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• 2006

The halophilic bacterium Vibrio vulnificus is known to be a foodborne pathogen that causes septicemia in human. V. vulnificus infection is characterized by the high fatality rates and the primary attack against a person who have underlying diseases such as liver cirrhosis. However, there is no effective treatment for V. vulnificus septicemia except for classical treatments such as antibiotics. Recently, it has been known that lipoprotein (LDL) plays a major role in the protection against infection and inflammation. Consequently in this paper we analyzed the effects of LDL on V. vulnificus septicemia. We purified V. vulnificus cytolysin, a major virulent factor of V. vulnificus infection and measured inhibitory effects of mouse serum, cholesterol, and LDL on its hemolytic activity. Next experiments were performed to investigate whether LDL has a protective role against septicemia induced by V. vulnificus in mice. Intraperitoneal injection of LDL (1mg as protein) into mice 3hr before V. vulnificus $(1\times10^6\;CFU)$ injection, and V. vulnificus -induced lethality was determined. For the determination the relationship between LDL or cholesterol and prognosis, we determined serum levels of cholesterol and lipoprotein from V. vulnificus septicemia patients (n=15) who had visited the Chonbuk National University Hospital in Chonju. V. vulnificus cytolysin -induced hemolysis of mice erythrocytes was completely inhibited by serum, cholesterol, and low-density lipoprotein. V. vulnificus- induced lethality of mice injected with LDL showed only 40% compared to 100% of control. In survival groups (n=4) of V. vulnificus septicemia patients (n=15), their serum LDL and cholesterol revealed normal levels ($153.3{\pm}40.7,\;LDL;\;190.8{\pm}16.3$, Total cholesterol). However, in death groups (n=11) showed very low levels ($35.6{\pm}13.9,\;LDL;\;59.2{\pm}15.1$, Total cholesterol). Our study indicates that cholesterol and LDL are a prognosis indicator of V. vulnificus septicemia as well as an inhibitor of virulent action of V. vulnificus cytolysin. We suggested that the serum levels of cholesterol or LDL would be major index in the treatment and prevention of V. vulnificus septicemia.

• Proceedings of the Microbiological Society of Korea Conference
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• 2006.05a
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• pp.32-34
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• 2006

• Journal of Pharmacopuncture
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• v.2 no.1 s.2
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• pp.73-91
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• 1999

This study was carried out to invastigate the researches of Snake Venom and snakes which used in treatment 1. The fist literature that used the snake for treatment is Shin Nong Ben Cao Jing 2. Composition of Snake Venom is consist of Enzymatic proteins ; Phospholipase A(A1-2), Protease, L-amino acid oxidase etc, and Non-enzymatic proteins ; Crotamine(Cytolysin), Proteolytic factor(Hematoxin), Crotoxin(Neurotoxin) etc. 3. Main toxins in Snake Venom are Hematoxin, Cytolysin, Neurotoxin and Cardiotoxin. Lethal dose 50 value of Agkistrodon brevicaudus is $45.87{\mu}g$/18g, Agkistrodon saxatilis is $10.28{\mu}g$/18g, Agkistrodon ussuriensis is $8.68{\mu}g$/18g, therefore Agkistrodon ussuriensis has strongist Snake Venom of all in Korea. 4. Pharmacological actions of Snake Venom are anticoagulation, thrombolytic function, hypotensor etc. 5. Systemic syndromes and signs after snakebite are Dizziness(25.7%), Vomitting(23.1%), Fever(22%), Visual disturbance(18%), Headache(17.7%) and Dyspnea(17.6%), etc. 6. Local syndrome and sign after snakebite is Discoloration(54.2%), Bleeding(20.2%), Bullae(10.7%), Skinulcer(10.8%), etc. 7. Pathological syndromes after snakebite are WBC increase, Urine protein, Urine sugar, Haematuria and elevation of S-GDT, S-GPT etc. These syndromes are leaded by Hematoxin and Cytolysin. 8. Complication signs after snakebite are Cellulitis, Gastritis, Lympoma, Abscess etc. 9. Common function of Viperidae(Agkistrodon acutus or Zaocys dhumnades etc) is expelling the wind(祛風), removing obstruction in the channels(通絡), antipastic function(止痙). And it is used in order to cure hemiparesis, hemiplegia, facial palsy and CVA disease, etc. 10. Using way of snake for medical treatment is various like Herbal alchol therapy, pill, powder and injection etc. The Study on the Snake Venom should be carried out continuously for using of medical treatment.