Role of divalent metal ions in serum complement activity of the American alligator (Alligator mississippiensis)

Treatment of alligator serum with different concentrations of EDTA resulted in a concentration-dependent inhibition of serum-mediated sheep red blood cell (SRBC) hemolysis. This inhibition of serum-dependent hemolysis was observed for other chelators of divalent metal ions, such as phosphate and citrate. Treatment of alligator serum with 5 mM EDTA completely inhibited SRBC hemolysis, which could be totally restored by the addition of 5 mM Ca(2+) or Mg(2+), but not Cu(2+) or Ba(2+). These data indicate a specific need for Ca(2+) and/or Mg(2+) in the serum-mediated hemolysis of SRBCs. Kinetic analyses revealed that the addition of 30 mM EDTA 1 min after incubation of SRBCs with serum resulted in only 30% inhibition of hemolytic activity. However, addition of EDTA as early as 3 min post-incubation resulted in complete SRBC hemolysis. Pretreatment of serum with EDTA inhibited the hemolytic activity, but the activity could be restored in a time-dependent manner by the addition of Ca(2+)or Mg(2+). These data indicate that, as in human serum, the need for divalent metal ions occurs early in the alligator serum complement cascade.     

Characterization of serum complement activity of saltwater (Crocodylus porosus) and freshwater (Crocodylus johnstoni) crocodiles

We employed a spectroscopic assay, based on the hemolysis of sheep red blood cells (SRBCs), to assess the innate immune function of saltwater and freshwater crocodiles in vitro. Incubation of serum from freshwater and saltwater crocodiles with SRBCs resulted in concentration-dependent increases in SRBC hemolysis. The hemolytic activity occurred rapidly, with detectable activity within 2 min and maximum activity at 20 min. These activities, in both crocodilian species, were heat sensitive, unaffected by 20 mM methylamine, and completely inhibited by low concentrations of EDTA, suggesting that the alternative serum complement cascade is responsible for the observed effects. The hemolytic activities of the sera were inhibited by other chelators of divalent metal ions, such as phosphate and citrate. The inhibition of SRBC hemolysis by EDTA could be completely restored by the addition of 10 mM Ca2+ or Mg2+, but not Ba2+, Cu2+ or Fe2+, indicating specificity for these metal ions. The serum complement activities of both crocodilians were temperature-dependent, with peak activities occurring at 25-30 degrees C and reduced activities below 25 degrees C and above 35 degrees C.     

Antibacterial properties of serum from the American alligator (Alligator mississippiensis)

Treatment of alligator (Alligator mississippiensis) and human serum samples with Escherichia coli resulted in a time- and concentration-dependent inhibition of bacterial proliferation. When inoculated with E. coli, alligator serum exhibited 10-fold lower bacterial survival rates after 1 h than human serum. In addition, the antibacterial spectrum of alligator serum was shown to be much broader than that of human serum, with growth inhibition occurring in 100% of bacterial strains tested (compared to only 35% for human serum). Additional results showed that the antimicrobial activities of alligator serum could be completely inhibited by preincubation with proteases, indicating the proteinaceous nature of the antimicrobial activities. Furthermore, incubation of alligator serum at 56 degrees C for 30 min (classical human serum complement inactivation conditions) obliterated all antimicrobial properties of the alligator serum. The antibacterial activities occurred relatively quickly in vitro, with significant activity occurring within 5 min of inoculation with E. coli and maximal activity at 20 min. Also, the antimicrobial activity exhibited temperature dependence, with a substantial decrease in activity below 15 degrees C. These data suggest that the antimicrobial properties of alligator serum may be due to an active serum complement system.     

Presence and characterization of complement-like activity in the amphioxus Branchiostoma belcheri tsingtauense

The humoral fluid of Branchiostoma belcheri tsingtauense was examined for the presence of complement-like activity. The humoral fluid showed hemolytic activity for rabbit erythrocytes and those from species representing mammals, birds, amphibians and fish, but not sensitized sheep erythrocytes. There was no relationship between phylogeny of the target erythrocytes and degree of hemolysis. The hemolytic activity was optimally assayed at 20 degrees C, at pH 7.5, and in the presence of 10 mM Mg2+. The hemolytic activity was Mg2+-dependent and heat-sensitive, and was abrogated by treatment with rabbit anti-human C3 serum, zymosan, methylamine, hydrazine, and phenylmethylenesulfonyl fluoride. In addition, Western blotting and titration by turbidimetric immunoassay (TIA) revealed that amphioxus humoral fluid contained C3 component, and its concentration is about 1.17 mg/ml, which is comparable to C3 concentration in human or dog sera. These suggest that the hemolytic activities displayed by amphioxus humoral fluid appear to represent the vertebrate complement system probably operating via the alternative pathway.     

Amoebacidal effects of serum from the American alligator (Alligator mississippiensis)

Treatment of axenic Naegleria gruberi cultures with alligator serum resulted in time-dependent amoebacidal activity, with measurable activity at 5 min and maximal activity occurring at 20 min. The amoebacidal activity was concentration dependent, with measurable activity at 25% serum, whereas treatment of amoebas with undiluted serum resulted in only 16% survival. The efficacy was dependent on the concentration of amoebas, with higher survival rates at high amoeba densities and lower survival rates at low amoeba densities. The amoeba-killing effects of alligator serum were broad in spectrum because the serum was effective against 3 strains of Naegleria species tested and 4 Acanthamoeba species, which have been reported to be resistant to human serum complement-mediated lysis. The amoebacidal effects of alligator serum were temperature dependent, with optimal activity at 15-30 C and a decrease in activity below 15 C and above 30 C. The amoebacidal activity of alligator serum was heat labile and protease sensitive, indicating the proteinaceous nature of the activity, and was also inhibited by ethylenediaminetetraacetic acid, which indicated a requirement for divalent metal ions. These characteristics strongly suggest that the amoebacidal properties of alligator serum are because of complement activity.     

Isolation and characterization of the third component of complement in the serum of the clawed frog, Xenopus laevis

The hemolytic activity against SRBC in the serum of normal Xenopus is dependent on specific antibody and both Ca++ and Mg++, whereas the activity against RRBC is dependent on Mg++ alone. Both of these hemolytic activities disappeared after treatment of the serum with zymosan or with the specific rabbit antiserum against one of the zymosan-binding proteins in Xenopus serum. By using this antiserum as a probe, a complement component (XC) was purified as a single entity from the Xenopus plasma after polyethylene glycol precipitation, DEAE-Sepharose CL-6B, Sepharose CL-6B, and Sephadex G-200 column chromatographies. The XC, contained at 2.3 mg/ml in normal serum, showed an electrophoretic mobility of beta-globulin, with a m.w. of 204,000 (204K) comprising two distinct subunits of 125K and 85K, which are linked with each other by disulfide bonds. The 204K protein exhibited a strong hemolytic activity in association with other components in Xenopus serum. Digestion of 204K protein by trypsin resulted in a specific cleavage of the 125K subunit and a conversion of its immunoelectrophoretic mobility to the anodal side, leaving the 85K subunits intact. The treatment of XC with SDS and urea resulted in the splitting of 125K subunits into 78K and 40K, but this splitting was inhibited upon pretreatment with methylamine, suggesting the presence of a thiol ester bond in the XC. The amino acid composition of the XC revealed a striking resemblance to that of mammalian C3. In all aspects, the 204K protein (XC) is regarded as representing the C3 of Xenopus laevis, which plays a key role in both the classical and alternative hemolytic pathways.     

荷斯坦种公牛血清替代补体溶血活性研究

本文将国外脊椎动物血清补体溶血活性标准测定方法,运用到荷斯坦种公牛研究中,首次建立了测定荷斯坦种公牛血清补体溶血ACH50的方法。种公牛血清经相应靶红细胞吸附后,可溶解悬浮在EGTAMgGVB缓冲液中的正常的兔血红细胞、人A,B,AB,O型红细胞,小鼠、大鼠、鸡红细胞,但对绵羊、山羊、猪红细胞溶血活性较低;对奶牛红细胞无溶血活性。且发现种公牛血清的溶血活性和靶红细胞的动物种类在系统发育上和种公牛的亲缘关系远近没有直接联系。种公牛血清在EGTAMgGVB缓冲液中对兔血红细胞发生溶血的最适条件是:温度是37℃,最适pH是7.3-7.4,最适Mg2 的浓度是4mmol/L,最适孵育时间为90min。溶血活性是二价离子依赖、热敏感(溶血活性热灭活温度是56℃)。种公牛血清对兔血红细胞的溶血活性在受到酵母聚糖、甲胺、肼、EDTA、鸡抗酵母聚糖牛血清结合物抗血清处理时,溶血活性可全部或部分消失,溶血活性抑制程度与补体抑制剂浓度相关。我们运用建立的标准溶血方法并以兔血红细胞作为指示细胞检测不同年龄的53头种公牛血清补体替代途径的溶血活性,溶血值在13.2-44.3u/ml之间,还发现不同年龄组公牛之间溶血活性有随年龄增加而逐步增大趋势,但差异不显著(P>0.05),在4-5岁公牛群中达到最大值。对种公牛血清补体系统溶血水平进行系统研究,一方面可以填补国内在此领域研究空白,另一方面也利于种公牛疾病监测、控制,此外也为兽医临床诊断试剂的研制提供新的技术手段。     

Trifluoperazine inhibits Sendai virus-induced hemolysis

Sendai virus-induced hemolysis, a manifestation of virus-red cell fusion, is inhibited by exposure of the virus to 50 microM and higher concentrations of trifluoperazine. Trifluoperazine does not disrupt the virus, since trifluoperazine-treated virus with no hemolytic activity sediments slightly faster than untreated virus on sucrose density gradients and contains viral proteins in proportions characteristic of untreated virus. Trifluoperazine affects the fusion protein to a greater extent than the hemagglutinin, since trifluoperazine-treated virus with no hemolytic activity is as active or nearly as active in agglutinating red cells. The partition coefficient of trifluoperazine between the virus membrane and buffer is lower at 4 degrees C than, but the same at 37 degrees C, as that between the red cell membrane and buffer. Nevertheless, virus-independent red cell lysis and inactivation of virus-mediated hemolysis occur when the red cell and viral membranes, respectively, contain similar concentrations of trifluoperazine. Furthermore, 13-28% more trifluoperazine is necessary to achieve either effect at 4 degrees C or at 25 degrees C than at 37 degrees C. Changes in the surface activity of trifluoperazine do not explain these results, insofar as the critical micellar concentration of (0.75 mM) and maximal reduction in surface tension by (40 dyn/cm) trifluoperazine are the same at 25 degrees C and 37 degrees C. The fluorescence of viral tryptophan decreases by approx. 25% when viral hemolysis is inactivated by trifluoperazine, by trypsin treatment or by heating at 100 degrees C for 5 min.     

Chemical and serological study of lipopolysaccharide isolated from Shigella flexneri 88-893 possessing a new type-antigen]

The O-specific polysaccharide chain which represents a new type-antigen in lipopolysaccharide (LPS) of Shigella flexneri 88-893 was investigated. The O-polysaccharide chain was found to be composed of repeating units comprising rhamnose, N-acetylglucosamine and glucose (3:1:2). In the passive hemolysis test, group-6 antiserum of S. flexneri exhibited a high hemolytic titer (50% hemolysis titer: 7,900) against sheep red blood cells (SRBC) sensitized with intact 893 LPS, but virtually no hemolytic activity against SRBC sensitized with alkali-treated 893 LPS. None of the type-specific antisera (I-VI), showed any significant hemolytic titer against SRBC sensitized with either intact or alkali-treated 893 LPS. Thus, 893 LPS contained both the group-6 antigen and a new type-antigen which is distinct from any known type-antigen of S. flexneri.     

Interactions between earthworm hemolysins and sheep red blood cell membranes

The hemolytic activity exhibited by the coelomic fluid of the Annelid Eisenia fetida andrei is mediated by two lipoproteins of mass 40 and 45 kDa, each of them capable of hemolysis. Such an activity is not inhibited by zymosan, inulin or lipopolysaccharide (LPS), nor by hydrazine or methylamine, suggesting that earthworm hemolysins are not related to C3 or C3b complement components. Among the membrane lipids tested (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin and cholesterol) only sphingomyelin inhibited hemolysis. The analysis of E.f. andrei proteins bound to sphingomyelin microvesicles, as well as to sheep red blood cell (SRBC) membranes, revealed a polymerization of E.f. andrei 40 kDa and/or 45 kDa hemolysins. Consequently, sphingomyelin appears a likely candidate for hemolytic complex receptor. Electron microscopy observations suggested that the polymerization causes an open channel through the lipid bilayer. As demonstrated using metal ions, heparin, chondroitin sulfate, poly(L-lysine) and protamine chloride, the mode of action of earthworm hemolytic complex is not analogous to that of C9 or perforine.     

Nonimmunologic complement activation in normal human serum induced by radiographic contrast media.

Two different radiographic contrast media (RCM), iothalamate and iodipamide, induced the activation of several complement (C) components in normal, genetically C2-deficient and agammaglobulinemic human sera in vitro. This activation was dose dependent and demonstrable by a reduction in whole C as well as C4, C2, C3, and C5 hemolytic activities. C6, C8, and C9 hemolytic activities were unaffected. Concommitant with the loss of C3 hemolytic activity was the appearance of C3 proteolytic cleavage products that were identified by immunoelectrophoresis. Both the loss of C3 hemolytic activity and the production of C3 fragments occurred in the presence of 10 mM EDTA, indicating RCM-induced C3 cleavage occurred without participation of the multicomponent C3/C5 convertases of either the classical or alternative C pathways. Furthermore, loss of C3 hemolytic activity was not due to the direct alteration of the C3 molecule by RCM because purified C3 was unaffected upon incubation with RCM at a concentration that induced 80% reduction in the C3 hemolytic activity in normal human serum. Serum samples obtained from 40 patients, before and 30 min after undergoing i.v. pyelography, revealed no significant change in total hemolytic C activity; 34 patients received sodium and methylglucamine diatrizoate and six received sodium iothalamate. Hemolytic C3 levels were also determined for the six patients before and 30 min after administration of sodium iothalamate and no significant change in activity was detectable.     

Purification of the sixth and seventh component of human complement without loss of hemolytic activity.

Procedures for the isolation of the human complement proteins C6 and C7 have been described. These procedures allow isolation of the two proteins without any loss of hemolytic activity. Apparent activity gains of 160% and 140% were observed for C6 and C7, respectively, when the activity of the isolated proteins was compared with their activity in serum. The recovery of C6 was 3.5 to 11% and that of C7 was 7 to 13% of the amount present in serum. C6 has a m.w.of 128,000 and an electrophoretic mobility at pH 8.6 of -2.6 times 10(-5) cm2 s-1 v-1. C7 has a m.w. of 121,000 and an identical electrophoretic mobility. With 3 times 10(7) assay cells, 63% hemolysis was achieved with 1 ng of C6 and 3.8 ng C7. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and after reduction with mercaptoethanol, C6 and C7 behaved as single polypeptide chain proteins.     

Association of activated properdin with complexes of properdin with C3

An ELISA using antibody to properdin (P), followed by antibody to C3 to detect complexes of P with C3 (P-C3), detected low levels of P-C3 complexes in human serum and plasma samples. Incubating serum for 1 h at 37 degrees C increased the amount of P-C3 and diminished factor B hemolytic activity without altering total alternative pathway activity or C3 activity in serum. When P and C3 in incubated serum were analyzed by size exclusion HPLC, complexes of P-C3 were detected at retention times corresponding to molecular mass measuring in excess of 2 x 10(6) Da. Activation of serum with zymosan or cobra venom factor greatly increased the level of P-C3 and decreased alternative pathway hemolytic activity. Chromatography of proteins eluted from serum-treated zymosan detected a peak of P at 9.7 x 10(5) Da and a peak of P-C3 at 1.5 x 10(6) Da. Functional assays for activated properdin also revealed a peak of activity at 1.5 x 10(6) Da, congruent with the peak of P-C3. Native properdin was detected at 3.9 x 10(5) Da. When native properdin was added to properdin-depleted serum and incubated for 1 h at 37 degrees C, activated properdin was detected at the same position in the chromatograph as were P-C3 complexes. We conclude that incubation of serum at 37 degrees C produces complexes of P with C3, that exposure of serum to alternative pathway activators increases the amount of P-C3, and that generation of P-C3 complexes is associated with the presence of activated P.     

Biochemical and pharmacological characterization of toxins obtained from the fire coral Millepora complanata

Millepora complanata is a normal resident of coral reefs in the Mexican Caribbean. In this study, we describe for the first time the vasoconstrictor, phospholipase A2 (PLA2), and hemolytic activities elicited by a crude extract obtained from M. complanata. This extract caused a concentration-dependent contraction of isolated rat aortic rings (EC50=22.4+/-1.1 microg protein/mL). This effect was endothelium independent and significantly reduced in the absence of extracellular Ca2+ and when the intracellular Ca2+ stores were depleted. In addition, the crude extract obtained from M. complanata showed PLA2 activity (7.231+/-0.092 mmol min(-1) mg(-1)) and hemolysis of rat erythrocytes (HU50=1.64+/-1.04 mug protein/mL). The hemolysis increased in the presence of Ca2+ and decreased in the presence of cholesterol. Furthermore, this hemolysis was significantly reduced after incubation with an inhibitor of PLA2 enzymes. The hemolytic and vasoconstrictor effects were abolished after incubating the extract under denaturing conditions. Reverse phase chromatography of the M. complanata extract afforded 19 fractions (F1 to F19). F4 induced hemolysis and contained mainly a protein of 30 kDa, probably a PLA2 enzyme, while F8 and F11, containing mainly proteins of 15 and 20 kDa respectively, produced vasoconstrictor effects mediated by different mechanisms of action.     

Variations in the responses of C57BL and a mice to sheep red blood cells: II. Analysis of plaque-forming cells

The number of direct and indirect plaque-forming cells (PFC) and the serum hemolytic activity was determined for A/He, C57BL/6J, and B6AF1 mice responding to multiple injections of sheep red blood cells (SRBC). Although the kinetics of the primary response differed, all mice had high numbers of both direct and indirect PFC and low-titered 2-mercaptoethanol (2-ME) sensitive serum antibody. Following multiple SRBC injections, the A/He spleens contained predominantly IgG producing PFC. Their serum antibody activity was resistant to 2-ME signifying the presence of IgG. The serum activity of both the C57BL/6J and B6AF1 mice was sensitive to 2-ME (IgM antibody) over the course of immunization, and although there was a definite IgM PFC memory response, the presence of 7S memory PFC was questionable. The results are discussed in terms of the maturation of the antibody response to SRBC and of the question of the postulated IgM and IgG switch.     

Purification and properties of the main coagulant and anticoagulant principles of Vipera russellii snake venom

Vipera russellii venom was separated into thirteen fractions by means of DEAE-Sephadex A-50 column chromatography. Fraction III possessed anticoagulant and phospholipase A activities and Fraction XI possessed procoagulant and caseinolytic activities, both were further purified by gel filtration on Sephacryl S-200 column. Purified procoagulant (Component II) was a two-chain protein with molecular weight of 86 000 consisting of A-chain (Mr 66 000) and B-chain (Mr 20 000). It was a glycoprotein containing 7.8% neutral sugar and 715 amino-acid residues. The procoagulant activity was 10-times that of the crude venom. It was an acidic proteinase with isoelectric point of pH 4.2. Upon heat treatment at 60 degrees C, Component II was stable at pH 5.5 and 7.2 for 3 h, but was destroyed completely after 30 min at pH 8.9. It was devoid of esterase or amidase activity. Purified anticoagulant (Component I) was a single peptide chain with molecular weight of 16 000. It was carbohydrate free and contained 136 amino-acid residues. It was a basic protein with an isoelectric point of larger than pH 10. It was a potent phospholipase A with an enzymatic activity of 510 +/- 30 mumol/min per mg using phosphatidylcholine as substrate, and 1 microgram/ml was sufficient to cause 100% hemolysis by the indirect hemolytic method. Upon heat treatment at 90 degrees C, Component I was heat stable at pH 5.5 for more than 3 h, but was destroyed completely after 2 h at pH 7.2 and 8.9. The anticoagulant activity of Component I could be neutralized by platelet factor 3, tissue thromboplastin and cephalin.     

Hemolysis of phosphatidylcholine-containing erythrocytes by serratamic acid from Serratia marcescens.

1. The hemolysis by serratamic acid, "N-(D-3-hydroxydecanoyl)-L-serine and N-(D-3-hydroxydodecanoyl)-L-serine", was investigated with human and animal erythrocytes using serratamic acid-containing liposomes. 2. The hemolytic activity was found to depend on the incubation temperature and the concentration of the liposomes. 3. The concentration of serratamic acid for 50% hemolysis was 0.17 mM at 37 degrees C for 0.2% human erythrocyte suspension in the liposomes which composed of phosphatidylserine, cholesteryl nervonate and serratamic acid (1:0.50:0.37 by mol). 4. The hemolysis was shown specifically in human, horse and rabbit erythrocytes containing phosphatidylcholine, but not in sheep or bovine erythrocytes lacking phosphatidylcholine. 5. The hemolytic activity was strongly inhibited by the exogenous addition of phosphatidylcholine. It was suggested that the hemolysis by serratamic acid-containing liposomes was specific for phosphatidylcholine-containing erythrocyte membranes.     

Mutational analysis of TlyA from Brachyspira hampsonii reveals two key residues conserved in pathogenic bacteria responsible for oligomerization and hemolytic activity

BackgroundTlyA proteins are expressed in a variety of pathogenic bacteria and possess dual hemolytic and ribosomal RNA methyltransferase functions. While the mechanism of TlyA mediated rRNA methylation is well understood, relatively little is known about the mechanism of TlyA induced hemolysis.MethodsTlyA protein from the pig pathogen Brachyspira hampsonii was heterologously expressed and purified from an E. coli host. Hemolytic activity and rRNA methylation were assessed in vitro. Site-directed mutagenesis was used to mutate amino acids believed to be involved in TlyA mediated hemolysis.ResultsPurified TlyA-His protein exhibited both hemolytic and rRNA methyltransferase activities in vitro, with partial inhibition of hemolysis observed under reducing conditions. Mutation of cysteine 80 to alanine impaired hemolytic activity. A C27A/C93A mutant was capable of dimerizing under non-reducing conditions, indicating that a C80-C80 disulfide bond is involved in TlyA oligomerization. A mutation conserved in several avirulent Brachyspira species (S9K) completely abolished hemolytic activity of TlyA. This loss of activity was attributed to impaired oligomerization in the S9K mutant, as assessed by ITC and size-exclusion chromatography experiments.ConclusionsOligomeric assembly and hemolytic activity of TlyA from Brachyspira hampsonii is dependent on the formation of an intermolecular C80-C80 disulfide bond and noncovalent interactions involving serine 9. The conservation of these amino acids in TlyA proteins from pathogenic bacteria suggests a correlation between tlyA gene mutations and bacterial virulence.General significanceOur results further elucidate the mechanisms underlying TlyA mediated hemolysis and provide evidence of a conserved mechanism of oligomerization for TlyA family proteins.     

Serum hemolytic activity in dogs infected with Babesia gibsoni

The hemolytic activity in serum of Babesia gibsoni-infected dogs was examined. When the activity was assayed in a reaction system consisting of similar concentrations of the serum and canine red blood cells to those in blood, significant hemolysis was observed. The activity of the serum of B. gibsoni-infected dogs, either naturally or experimentally, was always higher than that of uninfected animals. Moreover, in the experimental infection with B. gibsoni, the change in serum hemolytic activity was parallel to those of anemia and parasitemia, whereas it was inversely parallel to that of the hematocrit value. The present study revealed the presence of a hemolytic factor(s) in the serum of B. gibsoni-infected dogs, suggesting that the progressive anemia was due to hemolysis by the factor(s).