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ABSTRACT: Clostridium perfringens beta-toxin, an important agent of necrotic enteritis, causes plasma extravasation due to the release of a tachykinin NK(1) receptor agonist in mouse skin. In this study, we investigated the role of cytokines in beta-toxin-induced plasma extravasation.
Male Balb/c, C3H/HeN and C3H/HeJ mice were anaesthetized with pentobarbitone and beta-toxin was injected i.d. into shaved dorsal skin. SR140333, capsaicin, chlorpromazine and pentoxifylline were given as pretreatment when required before the injection of the toxin. Cytokines in the dorsal skin were measured by ELISA.
Injection (i.d.) of beta-toxin induced a dose-dependent increase in dermal TNF-alpha and interleukin (IL)-1beta levels with a concomitant increase in plasma extravasation, but not the release of IL-6. SR140333 and capsaicin significantly inhibited the toxin-induced release of TNF-alpha and IL-1beta. The plasma extravasation and the release of TNF-alpha induced by beta-toxin were significantly inhibited by chlorpromazine and pentoxifylline which inhibit the release of TNF-alpha. The toxin-induced plasma extravasation in mouse skin was attenuated by pretreatment with a monoclonal antibody against TNF-alpha, but not anti-IL-1beta. Furthermore, the toxin caused an increase in plasma extravasation in both C3H/HeN (TLR4-intact) and C3H/HeJ (TLR4-deficient) mice. In C3H/HeN mice, the toxin-induced leakage was not inhibited by pretreatment with anti-TLR4/MD-2 antibody.
These observations show that beta-toxin-induced plasma extravasation in mouse skin is related to the release of TNF-alpha via the mechanism involving tachykinin NK(1) receptors, but not via TLR4.
British Journal of Pharmacology 04/2008; 153(6):1296-302. · 4.41 Impact Factor
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ABSTRACT: Background and purpose:Clostridium perfringens beta-toxin, an important agent of necrotic enteritis, causes plasma extravasation due to the release of a tachykinin NK1 receptor agonist in mouse skin. In this study, we investigated the role of cytokines in beta-toxin-induced plasma extravasation.Experimental approach:Male Balb/c, C3H/HeN and C3H/HeJ mice were anaesthetized with pentobarbitone and beta-toxin was injected i.d. into shaved dorsal skin. SR140333, capsaicin, chlorpromazine and pentoxifylline were given as pretreatment when required before the injection of the toxin. Cytokines in the dorsal skin were measured by ELISA.Key results:Injection (i.d.) of beta-toxin induced a dose-dependent increase in dermal TNF-α and interleukin (IL)-1β levels with a concomitant increase in plasma extravasation, but not the release of IL-6. SR140333 and capsaicin significantly inhibited the toxin-induced release of TNF-α and IL-1β. The plasma extravasation and the release of TNF-α induced by beta-toxin were significantly inhibited by chlorpromazine and pentoxifylline which inhibit the release of TNF-α. The toxin-induced plasma extravasation in mouse skin was attenuated by pretreatment with a monoclonal antibody against TNF-α, but not anti-IL-1β. Furthermore, the toxin caused an increase in plasma extravasation in both C3H/HeN (TLR4-intact) and C3H/HeJ (TLR4-deficient) mice. In C3H/HeN mice, the toxin-induced leakage was not inhibited by pretreatment with anti-TLR4/MD-2 antibody.Conclusions and implications:These observations show that beta-toxin-induced plasma extravasation in mouse skin is related to the release of TNF-α via the mechanism involving tachykinin NK1 receptors, but not via TLR4.British Journal of Pharmacology (2008) 153, 1296–1302; doi:10.1038/bjp.2008.9; published online 11 February 2008
British Journal of Pharmacology 02/2008; 153(6):1296 - 1302. · 4.41 Impact Factor
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ABSTRACT: The iota(a) component (i(a)) of Clostridium perfringens ADP ribosylates nonmuscle beta/gamma actin and skeletal muscle alpha-actin. Replacement of Arg-295 in i(a) with alanine led to a complete loss of NAD(+)-glycohydrolase (NADase) and ADP-ribosyltransferase (ARTase); that of the residue with lysine caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them. Substitution of alanine for Glu-378 and Glu-380 caused a complete loss of NADase and ARTase. However, exchange of Glu-378 to aspartic acid or glutamine resulted in little effect on NADase activity but a drastic reduction in ARTase activity (<0.1% of the wild-type activity). Exchange of Glu-380 to aspartic acid caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them; that of the residue to glutamine caused a complete loss of ARTase activity. Replacement of Ser-338 with alanine resulted in 0.7 to 2.3% wild-type activities, and that of Ser-340 and Thr-339 caused a reduction in these activities of 5 to 30% wild-type activities. The kinetic analysis showed that Arg-295 and Ser-338 also play an important role in the binding of NAD(+) to i(a), that Arg-295, Glu-380, and Ser-338 play a crucial role in the catalytic rate of NADase activity, and that these three amino acid residues and Glu-378 are essential for ARTase activity. The effect of amino acid replacement in i(a) on ARTase activity was similar to that on lethal and cytotoxic activities, suggesting that lethal and cytotoxic activities in i(a) are dependent on ARTase activity.
Journal of Bacteriology 04/2000; 182(8):2096-103. · 3.83 Impact Factor
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ABSTRACT: Replacement of the Trp-1 in Clostridium perfringens alpha-toxin with tyrosine caused no effect on hemolytic and phospholipase C (PLC) activities or on binding to the zinc ion, but that of the residue with alanine, glycine and histidine led to drastic decreases in these activities and a significant reduction in binding to the zinc ion. The hemolytic and PLC activities of W1H and W1A were significantly increased by the preincubation of these variant toxins with zinc ions, but the preincubation of W1G with the metal ion caused little effect on these activities. Gly-Ile-alpha-toxin, which contained an additional Gly-Ile linked to the N-terminal amino acid of alpha-toxin, did not show hemolytic activity, but showed about 6% PLC activity of the wild-type toxin. A mutant toxin, which contained an additional Gly-Ile linked to the N-terminus of a protein lacking 4 N-terminal residues of alpha-toxin, showed about 1 and 6% hemolytic and PLC activities of the wild-type toxin, respectively. Incubation of the mutant toxin with zinc ions caused a significant increase in PLC activity. These observations suggested that Trp-1 is not essential for toxin activity, but plays a role in binding to zinc ions.
Microbiology and Immunology 02/2000; 44(7):585-9. · 1.30 Impact Factor
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ABSTRACT: A recombinant Ia component of Clostridium perfringens iota toxin, which ADP-ribosylates actin, was crystallized by the hanging drop vapor diffusion method using PEG4000 as a precipitating agent. The crystals were obtained in the presence of NADPH, which is similar to a real substrate, NADH, and belongs to the space group P1 (a = 47.9 A, b = 54.5 A, c = 103.1 A, alpha = 99.0 degrees, beta = 93.3 degrees, and gamma = 107.2 degrees ). The Matthews coefficient of native crystal was 2.7, assuming 2 mol/asymmetric unit. Native data were collected at 2.4-A resolution. The results from a heavy-atom search showed that lanthanide ions (samarium, holmium) altered the molecular packing, judging from the unit-cell difference. The crystals also belonged to the space group P1 (a = 47.7 A, b = 53.9 A, c = 54.6 A, alpha = 68.9 degrees, beta = 78.3 degrees, and gamma = 73.7 degrees ), which is consistent with only one molecule per asymmetric unit.
Journal of Structural Biology 07/1999; 126(2):175-7. · 3.41 Impact Factor
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ABSTRACT: The beta-toxin gene isolated from Clostridium perfringens type B was expressed as a glutathione S-transferase (GST) fusion gene in Escherichia coli. The purified GST-beta-toxin fusion protein from the E. coli transformant cells was not lethal. The N-terminal amino acid sequence of the recombinant beta-toxin (r toxin) isolated by thrombin cleavage of the fusion protein was G-S-N-D-I-G-K-T-T-T. Biological activities and molecular mass of r toxin were indistinguishable from those of native beta-toxin (n toxin) purified from C. perfringens type C. Replacement of Cys-265 with alanine or serine by site-directed mutagenesis resulted in little loss of the activity. Treatment of C265A with N-ethylmaleimide (NEM), which inactivated lethal activity of r toxin and n toxin, led to no loss of the activity. The substitution of tyrosine or histidine for Cys-265 significantly diminished lethal activity. In addition, treatment of C265H with ethoxyformic anhydride which specifically modifies histidyl residue resulted in significant decrease in lethal activity, but that of r toxin with the agent did not. These results showed that replacement of the cysteine residue at position 265 with amino acids with large size of side chain or introduction of functional groups in the position resulted in loss of lethal activity of the toxin. Replacement of Tyr-266, Leu-268 or Trp-275 resulted in complete loss of lethal activity. Simultaneous administration of r toxin and W275A led to a decrease in lethal activity of beta-toxin. These observations suggest that the site essential for the activity is close to the cysteine residue.
Biochimica et Biophysica Acta 06/1999; 1454(1):97-105. · 4.66 Impact Factor
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ABSTRACT: Clostridium perfringens epsilon-toxin bound to the Madin Darby canine kidney (MDCK) cells and aggregated. The complex of the toxin was formed in a dose- and a time-dependent manner. The formation of the complex increased with a decrease in viable counts of MDCK cells and with increasing K+ release from the cells. The inactivated toxin heated at 100 degrees C did not aggregate under the condition. In addition, the prototoxin dose-dependently bound to the cells, but did not form the complex. Incubation of the toxin with MDCK cell membranes also showed the formation of the complex, but that with membrane preparations prepared from Vero cells or sheep erythrocytes, which are insensitive for the toxin, showed no formation of the complex. Incubation of the toxin with mouse brain homogenates resulted in formation of the complex, but that with brain homogenates heated at 80 degrees C or mouse liver homogenates showed no formation of the complex. These observations show that the complex formation of epsilon-toxin is essential for toxicity of the toxin.
Journal of natural toxins 11/1998; 7(3):291-302.
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ABSTRACT: The effect of Clostridium perfringens alpha-toxin on liposomes prepared from phosphatidylcholine (PC) containing the fatty acyl residues of 18 carbon atoms was investigated. The toxin-induced carboxyfluorescein (CF) leakage and phosphorylcholine release from multilamellar liposomes increased as the phase transition temperature of the phosphatidylcholines containing unsaturated fatty acyl residues decreased. However, there was no difference between the sensitivity of the different phosphatidylcholines solubilized by deoxycholate to the phospholipase C (PLC) activity of the toxin. However, the toxin did not hydrolyze solubilized distearoyl-L-alpha-phosphatidylcholine (DSPC) or phosphatidylcholine containing saturated fatty acyl residue, and caused no effect on liposomes composed of DSPC. These results suggest that the activity of the toxin is closely related to the membrane fluidity and double bond in PC. The N-terminal domain of alpha-toxin (AT1-246) and variant H148G did not induce CF leakage from liposomes composed of dioleoyl-L-alpha-phosphatidylcholine (DOPC). H148G bound to the liposomes, but AT1-246 did not. However, the C-terminal domain (AT251-370) conferred binding to liposomes and the membrane-damaging activity on AT1-246. These observations suggest that the membrane-damaging action of alpha-toxin is due to the binding of the C-terminal domain of the toxin to the double bond in the PC in the bilayer and hydrolysis of the PC by the N-terminal domain.
Microbiology and Immunology 02/1998; 42(8):533-8. · 1.30 Impact Factor
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ABSTRACT: The current study has investigated the role of D-56, D-130, and E-152 in zinc ion binding properties, as well as the hemolytic, phospholipase C (PLC), and sphingomyelinase (SMase) activities of Clostridium perfringens alpha-toxin, based upon crystallography studies of the Bacillus cereus PLC, which had suggested these residues might be important for these functional activities. The replacement of D-56 in alpha-toxin resulted in complete loss of hemolytic, PLC, and SMase activities. The variant toxins at D-130 showed an approximately 100-fold reduction of biological activities compared to that of the wild-type toxin. The substitution of glutamine or glycine for E-152 caused complete loss of these activities, but substitution of aspartic acid for E-152 reduced but did not completely inhibit these activities. The variant toxins at D-56 and D-130, as well as the wild-type toxin, possessed approximately 2 mol of zinc atoms per mol of the protein, but E152G and E152Q contained approximately 1 mol of zinc metal per mol of the protein. On the other hand, the zinc content in E152D was calculated as about 1.4 mol in the toxin molecule. The replacement of D-56, D-130, or E-152 had no effect on binding to sheep erythrocytes and uptake of free zinc ion from the solution. The variant toxins at D-130 showed partial antigenic identity with the wild-type toxin on a double gel diffusion test. These observations suggest that D-56 in alpha-toxin is required for catalytic activity of alpha-toxin, D-130 is essential for maintenance of structure, and the carboxyl group of E-152 tightly ligands one zinc ion, which is essential for catalytic activity of the toxin.
Infection and Immunity 09/1997; 65(8):3489-92. · 4.16 Impact Factor
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ABSTRACT: Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.
FEMS Microbiology Letters 01/1997; 145(2):239-43. · 2.04 Impact Factor
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ABSTRACT: GTP and AIF4- significantly stimulated the late phosphatidic acid (PA) formation induced by Clostridium perfringens alpha-toxin in rabbit erythrocyte lysates. Pertussis toxin blocked the PA production. AIF4- markedly enhanced phosphatidylethanol production induced by alpha-toxin in the presence of ethanol. GTP[gamma S] stimulated the PA formation and hemolysis induced by alpha-toxin, and GDP[beta S] inhibited them. An H-to-G mutation at position 126 (H126G) induced the PA formation and hemolysis in a Co2+ concentration-dependent manner. H148G induced neither the PA formation nor hemolysis. These results suggest that the toxin-induced hemolysis is due to activation of phospholipid metabolism systems through GTP-binding protein.
Infection and Immunity 10/1996; 64(9):3930-3. · 4.16 Impact Factor
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ABSTRACT: The effect of Clostridium perfringens alpha-toxin on multilamellar liposomes prepared from various phospholipids and cholesterol was investigated. The toxin induced carboxyfluorescein leakage from liposomes composed of the choline-containing phospholipids such as egg-yolk phosphatidylcholine and bovine brain sphingomyelin in dose-dependent manner, but did not induce leakage from those liposomes composed of bovine brain phosphatidylethanolamine, egg-yolk phosphatidylserine or phosphatidylglycerol. The toxin-induced carboxyfluorescein leakage from egg-yolk phosphatidylcholine liposomes was increased by addition of divalent cations. The toxin induced carboxyfluorescein release from liposomes composed of phosphatidylcholine containing unsaturated fatty acyl residues or shorter chain length saturated fatty acyl residues (12 or 14 carbon atoms), but did not induce such release from liposomes composed of phosphatidylcholine containing saturated fatty acyl residues of between 16 and 20 carbon atoms. Furthermore, the toxin-induced carboxyfluorescein release decreased with increasing chain length of acyl residues of phosphatidylcholine used. The toxin bound to liposomes composed of phospholipids which are hydrolyzed by the toxin, but did not bind to those composed of phospholipids which are not attacked by the toxin. The toxin-induced carboxyfluorescein release from liposomes composed of dipalmitoleoyl-L-alpha-phosphatidylcholine and cholesterol and the toxin binding to the liposomes decreased with decreasing cholesterol contents. These observations suggest that the specific binding site formed by the choline-containing phospholipids and cholesterol, and membrane fluidity in liposomes are essential for the membrane-damaging activity of alpha-toxin.
Biochimica et Biophysica Acta 05/1996; 1280(1):120-6. · 4.66 Impact Factor
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ABSTRACT: A mutant toxin (MT) that abolished almost 99% of the hemolytic activity of alpha-toxin was isolated by random polymerase chain reaction (PCR) mutagenesis of the gene for Clostridium perfringens alpha-toxin. In the mutant toxin, the amino acids at Tyr (Y)-62, Thr (T)-74 and Ile (I)-345 were substituted with His, Ile and Met, respectively. Replacement of T-74 with Ile by site-directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250-fold of that of the wild-type. The replacement of Y-62 with Ile or I-345 with Met alone did not affect the activities of the toxin. T74I mutant bound to sheep erythrocyte membranes and specifically bound [65Zn]2+ in Tris-buffered saline, in the same manner as the wild-type, and contained 2 mol of zinc ions per mol of protein. These results suggest that the T-74 residue plays a key role in these biological activities of C. perfringens alpha-toxin.
Microbiology and Immunology 02/1996; 40(3):189-93. · 1.30 Impact Factor
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Advances in experimental medicine and biology 02/1996; 391:251-5. · 1.09 Impact Factor
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ABSTRACT: Mutagenesis of H-68 or -148 in Clostridium perfringens alpha-toxin resulted in complete loss of hemolytic, phospholipase C, sphingomyelinase, and lethal activities of the toxin. These activities of the variant toxin at H-126 or -136 decreased by approximately 100-fold of the activities of the wild-type toxin. Mutation at H-46, -207, -212, or -241 showed no effect on the biological activities, indicating that these residues are not essential for these activities. The variant toxin at H-11 was not detected in culture supernatant and in cells of the transformant carrying the variant toxin gene. Wild-type toxin and the variant toxin at H-148 bound to erythrocytes in the presence of Ca2+; however, the variant toxins at H-68, -126, and -136 did not. Co2+ and Mn2+ ions stimulated binding of the variant toxin at H-68, -126, and -136 to membranes in the presence of Ca2+ and caused an increase in hemolytic activity. Wild-type toxin and the variant toxins at H-68, -126, and -136 contained two zinc atoms in the molecule. Wild-type toxin inactivated by EDTA contained two zinc atoms. These results suggest that wild-type toxin contains two tightly bound zinc atoms which are not coordinated to H-68, -126, and -136. The variant toxin at H-148 possessed only one zinc atom. Wild-type toxin and the variant toxin at H-148 showed [65Zn]2+ binding, but the variant toxins at H-68, -126, and -136 did not. Furthermore, [65Zn]2+ binding to wild-type toxin was competitively inhibited by unlabeled Zn2+, Co2+, and Mn2+. These results suggest that H-68, -126, and -136 residues bind an exchangeable and labile metal which is important for binding to membranes and that H-148 tightly binds one zinc atom which is essential for the active site of alpha-toxin.
Journal of Bacteriology 04/1995; 177(5):1179-85. · 3.83 Impact Factor
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ABSTRACT: The effect of separate injections of two components of Clostridium perfringens iota toxin, designated Ia and Ib components, on the biological activities of the toxin was investigated. The intravenous injection of one component within 120 min after the injection of the other component killed mice. The activity of iota toxin was abolished by anti-Ia or anti-Ib antiserum. On the other hand, when Ib component was intravenously administered to mice given anti-Ia antiserum within 120 min after the intravenous injection of Ia component, the lethal activity was completely neutralized, but when Ia component was injected into mice that were given anti-Ib antiserum over 5 min after the injection of Ib component, the activity was not neutralized. The separate injections of Ia and Ib components in skin of guinea pigs indicated dermonecrosis at the injection site of Ib components, but not at the site of Ia components. Furthermore, when one component was intradermally injected in guinea pigs and then the other intraperitoneally, the dermonecrotic activity of the toxin was observed at the intradermal injection site of Ib component, but not at that of Ia component. From the data, it appears that the lethal and dermonecrotic activities of iota toxin are initiated by the binding of Ib component to specific sites on tissues.
Microbiology and Immunology 02/1995; 39(4):249-53. · 1.30 Impact Factor
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Nippon Saikingaku Zasshi 10/1994; 49(5-6):719-36.
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ABSTRACT: Treatment of Clostridium perfringens alpha toxin with aminopeptidase resulted in no effect on various activities of the toxin. Aminopeptidase did not hydrolyze the native toxin or the toxin treated with urea in the presence of EDTA. Treatment with carboxypeptidase for 30 min resulted in a 75% decrease in these activities. Incubation of the native toxin with carboxypeptidase for 30 min released approximately 15 amino acids from the C-terminus of the toxin. The biological activities of a mutant toxin lacking 20 C-terminal residues of the toxin (AT1-350) showed about 59-87% of the activity of native toxin. The mutant toxin showed partial antigenic identity with the native toxin. These data suggest that the C-terminal domain contributes to maintaining the active form of the toxin.
FEMS Microbiology Letters 08/1994; 120(3):297-301. · 2.04 Impact Factor
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ABSTRACT: The rapid phosphatidic acid (PA) formation induced by Clostridium perfringens alpha-toxin was stimulated by AlF4- in rabbit erythrocyte membranes. GTP[gamma S] [guanosine 5'-O-(3-thiotriphosphate)] stimulated the rapid 1,2-diacylglycerol formation and inositol 1,4,5-trisphosphate release induced by the toxin. On the other hand, treatment of erythrocyte lysates with phorbol 12-myristate 13-acetate (PMA) resulted in inhibition of toxin-induced PA production, and long-term PMA or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) treatment of the lysates led to stimulation of PA formation. Furthermore, treatment of erythrocytes with the toxin caused an increase of protein kinase C activity in membrane fractions. The results suggest that toxin-induced PA formation is mediated by endogenous phospholipase C regulated through GTP-binding protein and protein kinase C in rabbit erythrocytes.
Infection and Immunity 03/1994; 62(2):717-21. · 4.16 Impact Factor
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ABSTRACT: When rabbit erythrocytes were exposed to low concentrations of Clostridium perfringens alpha-toxin, hot-cold hemolysis was observed. The toxin induced production of phosphatidic acid (PA) in a dose-dependent manner when incubated with erythrocytes at 37 degrees C. When erythrocyte membranes were incubated with the toxin and [gamma-32P]ATP in the presence or absence of ethanol, [32P]PA formation was maximal within 30 s, then sharply decreased, and began again after 5 min of incubation. Ethanol had no effect on the early appearance (at approximately 5 min) of PA formation induced by the toxin but significantly inhibited formation of PA over 10 min of incubation. Treatment of erythrocyte membranes with alpha-toxin resulted in the biphasic formation of 1,2-diacylglycerol and PA as well as an increase of inositol-1,4,5-trisphosphate (IP3) and decrease of phosphatidylinositol-4,5-bisphosphate (PIP2) within 30 s. Neomycin inhibited the toxin-induced increase in turbidity of egg yolk suspensions but did not inhibit the toxin-induced hemolysis of intact erythrocytes. On the other hand, neomycin inhibited the toxin-induced hemolysis of saponin-treated erythrocytes. In addition, neomycin inhibited PA formation induced by the toxin in erythrocyte membranes. IP3 was released by incubation of PIP2 with erythrocyte membranes but not by incubation of PIP2 with the toxin. The toxin stimulated the membrane-induced release of IP3 from PIP2. These data suggest that the toxin-induced hemolysis is dependent on the action of phospholipase C in erythrocyte membranes.
Infection and Immunity 10/1993; 61(9):3711-8. · 4.16 Impact Factor
