Klebsiella pneumoniae haemolysin adsorption to red blood cells

It was necessary to incubate the Klebsiella pneumoniae haemolysin with erythrocytes at 37 degrees C to produce the whole lytic action. The amount of attached klebolysin at 4 degrees C increased as its concentration in the medium increased, until the erythrocyte surface was saturated. Treatment with 2-mercaptoethanol was necessary to permit the adsorption; it was inhibited by low concentrations of cholesterol. Klebsolysin was immunogenic and its antiserum neutralized its own haemolytic effect and streptolysin O. Anti-streptolysin serum also neutralized klebsolysin. Streptolysin O attachment to erythrocytes impeded the posterior klebolysin adsorption in the same way that klebsolysin adsorption interfered with streptolysin O attachment.     

Sterol structural requirements for inhibition of streptolysin O activity

Reduced streptolysin O, a toxin produced by certain beta-haemolytic streptococci, lyses human erythrocytes. The reaction is inhibited by cholesterol at concentrations of about 1.0mug/ml. Other sterols inhibit the lysin and there is a specific requirement for a 3beta-hydroxyl group. Inhibition was obtained with 3beta-hydroxychol-5-en-24-oic acid, containing a hydrophilic group at C-24. The mode of inhibition is likely to involve attachment to the fixation site of the lysin which attaches the molecule to cell membranes, probably to membrane cholesterol. A second streptolysin site, concerned in the final haemolytic event, may also be involved. Inhibitors of the latter site have not been characterized, other than antibody with specificity for the site.     

Detection of C-reactive protein, streptolysin O, and anti-streptolysin O antibodies in immune complexes isolated from the sera of patients with acute rheumatic fever

Circulating immune complexes (IC) of 42 patients with acute rheumatic fever from Santiago, Chile, were studied. The complexes were isolated by polyethylene glycol precipitation and were analyzed for antibodies, antigens, and C-reactive protein. We found the complexes to be enriched in antibody to streptolysin O, particularly in the group of patients with elevated levels of IC. IgM was the predominant class of Ig present in the complexes. Western blots from 12 patients to detect antigens in the complexes showed proteins of m.w. 50,000, 60,000, and 69,000, consistent with the polypeptides of streptolysin O. Such antigens were absent in the complexes from patients with post-streptococcal glomerulonephritis and pharyngitis. Eluted antibodies from these protein bands on the nitrocellulose sheets reacted with the streptolysin O in Western blots and neutralized the hemolytic activity of streptolysin O in a microhemolysin assay. In addition, isolated complexes from several sera showed the presence of C-reactive protein bound to complexes. In vitro experiments demonstrated that [125I]C-reactive protein was not precipitated by polyethylene glycol either alone or when added to monomeric IgG, whereas it precipitated significantly when added to aggregated IgG. The detectable C-reactive protein in isolated complexes and sera samples increased after treatment with sodium dodecyl sulfate. These data suggest that circulating immune complexes in acute rheumatic fever contain streptolysin O and its antibody and raise interesting questions regarding the pathogenetic significance of C-reactive protein in the complexes.     

COMPARATIVE KINETICS OF HEMOLYSIS INDUCED BY BACTERIAL AND OTHER HEMOLYSINS

A study has been made of the kinetics of lysis induced by various hemolytic agents. The course of bemolysis was followed by mixing lysin with washed human erythrocytes, removing samples from the mixture, and estimating colorimetrically the hemoglobin in the supernatant fluid of the centrifuged samples. Most of the curves (but not all of them, e.g. tyrocidine) obtained by plotting degree of hemolysis against time, were S-shaped. The initiation of lysis by streptolysin S'' was delayed, and in this property, streptolysin S'' was similar to Cl. septicum hemolysin. None of the other lysins studied exhibited a long latent period preceding lysis. The maximum rate of hemoglobin liberation was found, in the range of lysin concentrations studied, to be a linear function of concentration when theta toxin of Cl. welchii, pneumolysin, tetanolysin, or streptolysin S'' was the lytic agent. With comparable concentrations of saponin, sodium taurocholate, cetyl pyridinium chloride, tyrocidine, duponol C, lecithin-atrox venom mixture, or streptolysin O, the relation between rate and concentration was non-linear. The critical thermal increment associated with hemolysis was determined for systems containing pneumolysin, theta toxin, streptolysin S'', streptolysin O, tetanolysin, and saponin. The findings concerning the effect of concentration and temperature on the rate of hemolysis provide a basis for classifying hemolytic agents (Tables I and II). Hemolysis induced by Cl. septicum hemolysin was found to be preceded by two phases: a phase of alteration of the erythrocytes and a phase involving swelling. Antihemolytic serum inhibited the first but not the second phase while sucrose inhibited the second but not the first phase.     

Haematological changes by splenic respiratory compensation in the cave salamander, Hydromantes genei

Like other amphibians, the salamander Hydromantes genei , an exclusively terrestrial lungless plethodontid which lives in cold humid caves, possesses a haematological mechanism of respiratory compensation: the spleen can hoard erythrocytes, which are then released into the bloodstream when necessary, analogous to what happens in the Italian crested newt (which, however, is predominantly aquatic). The cave salamander, anaesthetized with chlorobutanol and kept in a humidity-saturated environment at a constant temperature long enough for its haematological conditions to stabilize, presents homogeneous and very low blood parameters at the extreme temperature ranges to which it is adapted (6 °C and 18 °C): about 16 10⁹ red blood cells/1, haematocrit value approximately 12, and haemoglobin concentration slightly below 3g/dl. The increase in temperature triggers the release of erythrocytes into the bloodstream from the spleen, which shrinks from 0.8% of the animal's body weight at 6 °C to 0.25% at 18 °C; however, a parallel increase in blood plasma maintains the blood composition unaltered. At 24 °C, a critical temperature for this species, the erythrocyte parameters increase by 50% owing to plasma loss, as happens in other amphibians in hypoxic conditions.     

Generation of a membrane-bound, oligomerized pre-pore complex is necessary for pore formation by Clostridium septicum alpha toxin

Low-temperature inhibition of the cytolytic activity of alpha toxin has facilitated the identification of an important step in the cytolytic mechanism of this toxin. When alpha toxin-dependent haemolysis was measured on erythrocytes at various temperatures it was clear that at temperatures ≤15°C the haemolysis rate was significantly inhibited with little or no haemolysis occurring at 4°C. Alpha toxin appeared to bind to and oligomerize on erythrocyte membranes with similar kinetics at 4°C and 37°C. The slight differences in these two processes at 4°C and 37°C could not account for the loss of cytolytic activity at low temperature. At 4°C alpha toxin neither stimulated potassium release from erythrocytes nor formed pores in planar membranes. In contrast, at temperatures ≥25°C both processes proceeded rapidly. Pores that were opened in osmotically stabilized erythrocytes could not be closed by low temperature. Therefore, low temperature appeared to prevent the oligomerized complex from forming a pore in the membrane. These data support the hypothesis that alpha toxin oligomerizes into a membrane-bound, pre-pore complex prior to formation of a pore in a lipid bilayer.     

Effect of streptolysin O on erythrocyte membranes, liposomes, and lipid dispersions. A protein-cholesterol interaction

The effect of the bacterial cytolytic toxin, streptolysin O (SLO), on rabbit erythrocyte membranes, liposomes, and lipid dispersions was examined. SLO produced no gross alterations in the major erythrocyte membrane proteins or lipids. However, when erythrocytes were treated with SLO and examined by electron microscopy, rings and "C"-shaped structures were observed in the cell membrane. The rings had an electron-dense center, 24 nm in diameter, and the overall diameter of the structure was 38 nm. Ring formation also occurred when erythrocyte membranes were fixed with glutaraldehyde and OsO4 before the addition of toxin. In contrast, rings were not seen when erythrocytes were treated with toxin at 0 degrees C, indicating that adsorption of SLO to the membrane is not sufficient for ring formation since toxin is known to bind to erythrocytes at that temperature. The ring structures were present on lecithin-cholesterol-dicetylphosphate liposomes after SLO treatment, but there was no release of the trapped, internal markers, K2CrO4 or glucose. The crucial role of cholesterol in the formation of rings and C's was demonstrated by the fact that these structures were present in toxin-treated cholesterol dispersions, but not in lecithin-dicetylphosphate dispersions nor in the SLO preparations alone. The importance of cholesterol was also shown by the finding that no rings were present in membranes or cholesterol dispersions which had been treated with digitonin before SLO was added. Although rings do not appear to be "holes" in the membrane, a model is proposed which suggests that cholesterol molecules are sequestered during ring and C-structure formation, and that this process plays a role in SLO-induced hemolysis.     

Effect of temperature and dissolved oxygen concentration on the metabolic rate of the turbot and the relationship between metabolic scope and feeding demand

Turbot Scophthalmus maximus maximum oxygen uptake following feeding and exhaustive exercise increased from 107 mg O2 kg⁻¹ h⁻¹ at 6° C to c . 218 mg O2 kg⁻¹ h⁻¹ at 18° C, then increased slightly from 18 to 22° C to 224 mg O2 kg⁻¹ h⁻¹. Standard oxygen uptake increased exponentially as a function of temperature from 11 mg O2 kg ⁻¹ h⁻¹ at 6° C to 66 mg O2 kg⁻¹ h⁻¹ at 22° C. Gradual reduction in oxygen concentration to 87–90% air saturation at 6, 10. 18° C and <80% at 14 and 22° C limited the maximum metabolic rate but, supersaturation (>100% saturation) had little effect. Metabolic scope attained a maximum of 176 mg O₂ kg⁻¹ h⁻¹ at 18° C. Interpolation of the results showed that this value changed little between 16 and 20° C. It is suggested that this temperature range is optimal for turbot of c . 500 g. A comparison with a previous study on feeding demand in intensive farming conditions showed a linear relationship between appetite and metabolic scope. It is concluded that the ability of a fish to supply energy (including the energy requirement of digestive metabolism) above a standard level is a limiting factor in the manifestation of its feeding demand.     

Extracellular Hemolysins of Aerobic Sporogenic Bacilli

Forty-five strains, representing 18 species of the genus Bacillus, were surveyed for production of hemolysin against rabbit erythrocytes. Broth cultures of B. cereus, B. alvei, and B. laterosporus contained lysins that closely resembled streptolysin O. B. subtilis and a single strain of B. cereus may produce lysins having characteristics different from those of streptolysin O.     

Site selection and attachment duration of Anodonta woodiana (Bivalvia: Unionacea) glochidia on fish hosts

The relationship between parasitic glochidia of Anodonia woodiana (Unionidae: Anodonti-nae) and potential fish hosts was investigated in the laboratory. Intensity of parasitism was highest on the exotic fish Gambusia affinis , lower on the native species Puntius semijascio-latus and Metzia takakii , and least on Rhodeus sinensis . Glochidia generally attached at fin margins, particularly the pectorals and caudal. In Gambusia affinis , the incidence of glochidia on the pectorals was higher than would be expected on the basis of ratios between fin margin lengths. Apparently, the role of the pectoral fins in locomotion makes them more liable to glochidial contact, thereby increasing their susceptibility to attachment.
The duration of glochidial attachment was shortened as temperatures increased. Mean values ranged from 14-4 days at 15°C to 6 days at 27°C. At 33°C glochidia rapidly detached and metamorphosis was unsuccessful. Significantly, water temperatures in A. woodiana habitats in Hong Kong rarely exceed 30°C.     

Gametocytogenesis of Leucocytozoon caulleryi in In Vitro Culture: Effect of Human Red Blood Cells on the Development of Second-Generation Merozoites

For investigating development of second-generation merozoites of Leucocytozoon caulleryi into mature gametocytes, infected erythrocytes from chickens at 15 d after sporozoites inoculation were cultured in RPMI-1640 modified medium supplemented with 10% horse serum and 0.5 ml of human erythrocytes (type O). When culture was carried out at 37°C in a humidified atmosphere of 5% CO₂ for 7 d, the very small number of second-generation merozoites developed into morphologically mature gametocytes. However, in the high carbon dioxide and low oxygen condition, mature gametocytes weren't observed in cluture. The role of human erythrocytes added has not been clarified yet.     

Studies on the mechanism of action of oxygen-labile haemolysins.

The sensitivities of the binding step and the lytic step of haemolysis by pneumolysin to the action of various inhibitors and to variations in the assay conditions were studied. Binding was inhibited by HgCl2 and N-ethylmaleimide. Lysis by previously fixed lysin was insensitive to HgCl2 and only slightly sensitive to N-ethylmaleimide. Binding of pneumolysin was independent of ionic strength. Binding of pneumolysin and streptolysin O decreased above pH 8-0 and 8-4 respectively. These results suggest that binding requires a non-ionized unsubstituted sulphydryl group. Incubation of erythrocytes with NaF caused inhibition of pneumolysin, indicating that some metabolic function of the cell may be involved in lysis. The action of streptolysin O was not affected by NaF.     

Time Sequence of Early Events in Fertilization in the Medaka Egg

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes . The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 10⁸/ml at 23°–25°C). The mean time from sperm attachment until cessation of its movement on the egg surface was 4 ± 1 sec with a range from 1 to 9 sec. Small cortical alveoli at the animal pole region within 15 μm of the sperm attachment point began to undergo exocytosis 9 ± 0.3 sec (range 5–16 sec) after sperm attachment. The velocity at which the exocytosis wave propagated increased from the earliest initiation point of exocytosis up to the 100 μm area, and became constant at about 12 μm/sec from 100 μm to 500 μm from the sperm attachment point. The present results suggest that at the time of fertilization in the fish egg, exocytosis of small cortical alveoli in the area about 15 μm away from the sperm attachment point occurs simultaneously.     

Interaction of steptolysin O with sterols.

A quantitative study of the specific inhibitory power of cholesterol and other sterols on the hemolytic properties of streptolysin O is reported. This streptococcal exocellular protein is a cytolytic toxin which disrupts cytoplasmic membranes of eukaryote cells. The structural characteristics, particularly the stereochemical ones required for a steroid molecule to inhibit the cytolytic activity of streptolysin O, have been investigated in detail. By immunodiffusion techniques, in agar gel plates or tubes containing sterols, the formation of hydrophobic complexes between streptolysin O and inhibitory steroids, but not non-inhibitory steroids except lanosterol, is shown. Upon interaction with inhibitory steroids streptolysin O loses its immunoreactive properties towards neutralizing and precipitating homologous antibodies. An interpretation of the mechanism of biomembrane disorganization by streptolysin O is discussed in the light of its steroid binding properties.     

Storage of marine fish erythrocytes in liquid nitrogen

The preservation of erythrocytes from cod ( Gadus morhua ), saithe ( Pollachius virens ) and mackerel ( Scomber scombrus ) at −196° C was studied using dimethyl sulphoxide (DMSO) as a cryoprotectant. Erythrocyte recoveries of greater than 90% were obtained from all species and cod erythrocytes were stored for eighteen months with insignificant lysis. Larger quantities of blood were stored by removal of plasma from citrated blood prior to the addition of DMSO solution, and by storage of pelleted frozen blood in aluminium canisters in liquid nitrogen. Maximum recoveries of washed intact erythrocytes required thawing of pellets in 125% DMSO solution and washing with buffer containing decreasing concentrations of DMSO. Washed erythrocytes kept at 4° for at least two days showed little haemolysis, were morphologically similar to fresh erythrocytes and equally susceptible to the δ-haemolysin of Staphylococcus aureus .     

Differential effects of lectins mediating erythrocyte attachment and ingestion by macrophages

Lectin-mediated interaction of erythrocytes and macrophages was brought about in two steps. Step I involved macrophage treatment with lectin, and step II is the incubation of lectin-treated macrophages with mouse erythrocytes. The extent and nature of lectin-mediated macrophage erythrocyte interaction was studied using concanavalin A (ConA), wheat germ (WGA), soybean (SBA) and waxbean (WBA) agglutinins. The parameters affecting the interaction were studied in detail with the first two lectins.Under comparable conditions of lectin interaction with macrophages (step I), WGA mediates rosette formation involving interaction with several times the number of erythrocytes than those interacting with ConA-treated macrophages. The interaction mediated by WGA reaches, at 37 °C, a saturation value after 30 min of step II, whereas that mediated by ConA is still linear and exhibits half the amount of attached erythrocytes at 60 min. ConA-mediated attachment of erythrocytes is highly temperature-dependent being at 37 °C twice that observed at 24 °C. The temperature dependence of attachment is not affected by changes of either ConA concentration (5–40 μg/ml) or the temperature in step I. An optimum is observed, however, when the temperature of incubation in step I ranges between 14–18 °C. WGA-mediated attachment of erythrocytes is markedly less temperature-sensitive, exhibiting 70% of optimal attachment already at 8 °C. Only when the attachment phase follows incubation with a low concentration of WGA (2 μg/ml) high temperature sensitivity is exhibited. At 37 °C, however, the number of attached erythrocytes is the same for macrophages treated with WGA at concentrations of 2, 5, 10 and 40 μg/ml.ConA-mediated erythrocyte-macrophage interaction does not lead to erythrophagocytosis. When mediated by WGA, the attachment step is followed by a temperature-dependent ingestion step, i.e. 10% and 50% of the erythrocytes that attach to macrophages during the 60 min incubation at 24 °C and 37 °C, respectively, are ingested. There is a lag period of 10–20 min between attachment and ingestion implicating involvement of additional cellular processes preceding engulfment. Electron microscope images of areas of interaction of attached erythrocytes with macrophages indicate a significantly tighter binding (a thinner gap at membrane-membrane apposition areas) in the case of WGA-mediated rosette formation as compared with that established in ConA-mediated rosettes. Attachment via WGA is followed by a rapid change in the relative position of the attached erythrocytes on the macrophage, from a primary attachment at the distal peripheral regions of the cell, to a perinuclear position. In contrast, erythrocytes attached via ConA remain at the primary attachment point (at 37 °C) for extended periods. This differential behaviour does not stem from effects of ConA on macrophages, since when yeast cells were attached to ConA treated macrophages, the yeast cells showed the same movement as that exhibited by erythrocyte when attached via WGA.The different interaction patterns of erythrocytes with macrophages coated with ConA and WGA can be fitted into the following working hypothesis: the number of WGA-binding sites on the plasma membrane of macrophages is at least three times that of ConA-binding sites. Stable cell-cell interactions involve multibridge formation at the contact area of the two cells and this involves a delicate balance between number of lectin-receptor conjugates and their aggregation state within the membrane phase. A certain amount of clustering is a prerequisite for attachment, while a high degree of clustering reduces the chance of fruitful interactions. The engulfment step depends on the ability of membrane areas adjacent to primary contact area to establish additional stable bridges in the entire circumference of the attached cell. ConA-receptor conjugates appear to be less abundant and more aggregated within the membrane plane, preventing the completion of fruitful circumferential interaction of the adjacent membrane. WGA-receptor conjugates, being more abundant and apparently less aggregated are available at membrane areas needed for cell enclosure and provide the additional bridging without which engulfment does not take place. Change in relative position of attached erythrocytes seems to be a step in the manifold events occurring from attachment to ingestion.     

Splenic respiratory compensation and blood volume in the newt

In the newt Triturus cristatus carnifex , reversible increase in size of the spleen is linked to the respiratory state of the animal: when the newt is exposed to the air, and thus well oxygenated, the spleen hoards erythrocytes; when immersed in still water, in an hypoxic state, the spleen releases erythrocytes into the bloodstream. In chlorobutanol-anaesthetized specimens exposed to the air, the maximum size reached by the spleen diminishes with a rise in temperature up to the disappearance of all congestion at 33°C. The blood volume of newts kept in humid air at 6°C and 18°C after anaesthesia varies from about 6–9 ml per 100 g of body weight, while the red blood cell count and haematocrit value remain stable. In anaesthetized specimens kept in still water at the same temperatures the blood volume is stable, at about 7 ml per 100 g, but the red cell count and haematocrit are notably higher. At 33°C, a critical temperature for the newts, the specimens in still water succumb while those in air present the same blood volume as at 18°C, but have a higher erythrocyte count and haematocrit value.     

Rapid small scale preparation of bacterial genomic DNA, suitable for cloning and hybridization analysis

The uptake of noxythiolin by a urinary isolate of Escherichia coli was examined initially at 37°C but the adsorption isotherm was complicated by the concomitant degradation of the compound. When drug adsorption was investigated at 4°C, to reduce the degradation rate of the compound, it was observed that noxythiolin was taken up by the urinary isolate in a linear fashion. The resulting adsorption patterns are discussed in relation to their possible classification. The implications of this uptake are considered with respect to the antimicrobial activity of noxythiolin.     

Internode length in Pisum. I. The effect of the Le/le gene difference on endogenous gibberellin-like substances

The allelopathic potential of the dry fruits of Washingtonia filifera (L. Linden) H. Wendl. was investigated. Leachates from fruits inhibited the germination of lettuce, wheat, red cabbage and cucumber seeds. The inhibitory effect was partly neutralized by kinetin (20 mg 1⁻¹) and gibberellic acid (50 mg 1⁻¹). The effect of kinetin was more pronounced at 25°C than at 20°C. Substances inhibiting germination were localized in the pericarp of the fruit and were resistant to high temperature.     

Selective toxin-lipid membrane interactions of natural, haemolytic Scyphozoan toxins analyzed by surface plasmon resonance

A comparison of the molecular interaction of natural Scyphozoan lysins with their bioactivity in a haemolytic assay was performed by establishing an efficient, automatable and reproducible procedure for the measurement of protein-membrane interactions. The toxin-membrane interactions were analyzed utilising a chip-based technology with immobilized liposomes as artificial cell membranes. The technique was established with streptolysin O as a cholesterol-selective model toxin and its cholesterol-selectivity has been proven. The haemolytic potency of protein fractions derived from the venom of the jellyfish Aurelia aurita and Cyanea capillata was tested and EC50 values of 35.3 μg/mL and 43.1 μg/mL against sheep and 13.5 μg/mL and 8.8 μg/mL against rabbit erythrocytes were measured. Cell membrane binding as a first step in the haemolytic process was analyzed using the Biacore^® technology. Major cell membrane lipids (cholesterol, sphingomyelin and phosphatidylcholine) were immobilized as pure liposomes and in binary mixtures. A preference for cholesterol and sphingomyelin of both jellyfish species was demonstrated. The specificity of the method was proven with a non-haemolytic A. aurita protein fraction that did not express a lipid binding. Additionally, an inactivated C. capillata lysine with negligible haemolytic activity showed a remaining but reduced adsorption onto lipid layers. The binding level of the lytic venom fraction of these dominant boreal jellyfish species increased as a function of protein concentration. The binding strength was expressed in RU50 values ranging from 12.4 μg/mL to 35.4 μg/mL, which were in the same order of magnitude as the EC50 values in the haemolytic assay.