Activation of the calcium permeability of erythrocyte membranes by perfringolysin O

Calcium ions inhibited perfringolysin O-induced hemolysis at a concentration lower than 1 mM, but not the hemolysis by digitonin at 10 mM. The introduction of calcium ions into ghosts inhibited the lysis more strongly than the addition of calcium ions outside ghosts. When erythrocytes were treated with perfringolysin O in the presence of 1 mM CaCl2 containing 45CaCl2, the radioactivities inside cells rapidly increased during incubation. On the other hand, when perfringolysin O-treated erythrocytes were incubated in a calcium-free medium, the erythrocytes released calcium ions at a 3.3-fold higher rate than untreated cells. These results suggested that perfringolysin O accelerated both the calcium influx into and efflux from erythrocytes.     

Bacteriocin (hemolysin) of Streptococcus zymogenes

The sensitivity of Streptococcus faecalis (ATTC 8043) to S. zymogenes X-14 bacteriocin depends greatly on its physiological age. Sensitivity decreases from the mid-log phase on and is completely lost in the stationary phase. The sensitivity of erythrocytes to the hemolytic capacity of the bacteriocin showed considerable species variation. The order of increasing sensitivity was goose < sheep < dog < horse < human < rabbit. However, when red cell stromata were used as inhibitors of hemolysis in a standard system employing rabbit erythrocytes the order of increasing effectiveness was sheep < rabbit < human < horse < goose. When rabbit cells were used in varying concentrations with a constant hemolysin concentration, there was a lag of about 30 min, which for a given hemolysin preparation was constant for all red cell concentrations. Furthermore, the rate of hemolysis increased with increasing red cell concentration. If red cells are held constant and lysin varied, the time to reach half-maximal lysis varies directly with lysin but is not strictly proportional. Bacterial membranes were one to three orders of magnitude more effective than red cell stromata as inhibitors. The order of increasing effectiveness seems to be Escherichia coli < Bacillus megaterium < S. faecalis < Micrococcus lysodeikticus. In addition to membranes, a d-alanine containing glycerol teichoic acid, trypsin in high concentration, and deoxyribonuclease also inhibited hemolysis. Ribonuclease, d-alanine, l-alanine, dl-alanyl-dl-alanine, N-acetyl-d-alanine, N-acetyl-l-alanine did not inhibit hemolysis.     

Viscous macromolecules inhibit erythrocyte hemolysis induced by snake venom phospholipase A2

The effect of medium viscosity on lysis of red blood cells (RBC) induced by snake venom phospholipase A2 (PLA2) was examined. The medium viscosity was modified by the addition of various macromolecules which differ in their chemical nature and in their capacity to increase fluid viscosity. PLA2 and Ca++ were applied to cells suspended in viscous medium to induce hemolysis. It was found that the hemolysis is inhibited in direct proportion to increasing viscosity of the extracellular fluid. This phenomenon was observed with aggregated as well as disaggregated RBC. To examine whether the viscosity interferes with the accessibility of the enzyme to the cell, the medium viscosity was modified after binding of the enzyme to the cells; PLA2 was added to a RBC suspension in the presence of Ba++ which binds the enzyme to the cell membrane but does not activate it. The cell-enzyme complex was separated by gel filtration and suspended in viscous medium in the presence of Ca++ which activates the reaction. Also in this case RBC lysis was inhibited as the medium viscosity was increased. It is proposed that the action of PLA2 on RBC membrane is regulated by the viscosity of the cell surface aqueous environment.     

A zone phenomenon and a progressive reaction occurring with a radioactive hemolysin,sodium erucate,I 131

Sodium erucate reacts progressively (i.e., once the reaction is started in a time which is so short that the lysin is in contact with the red cells for 30 seconds, it cannot be stopped even by being diluted 10-fold) with human red cells at pH 7. At the same time, systems containing the lysin and human red cells show a zone phenomenon, lysis occurring most readily in a certain concentration of lysin but more slowly in larger or smaller concentrations. Sodium erucate-I¹³¹ can be used to investigate both the zone phenomenon and the progressive character of the reaction. As regards the former, large concentrations of the lysin react relatively poorly with the red cell surfaces and the resistance of the red cells is relatively high. This may be due to the presence of an admixed inhibitor or to the development of an inhibitory state. The lysin is taken up and fixed by material in the red cell surface, so that the "internal phase" of lysin attached to the cell surfaces is so firmly fixed that a 10-fold dilution has no effect on it. It follows that lysis in these systems is progressive, as it is found to be.     

Temperature‐dependent hemolytic activity of membrane pore‐forming peptide toxin,tolaasin

Tolaasin, a pore‐forming peptide toxin produced by Pseudomonas tolaasii, causes brown blotch disease on cultivated mushrooms. Hemolysis using red blood cells was measured to evaluate the cytotoxicity of tolaasin. To investigate the mechanism of tolaasin‐induced cell disruption, we studied the effect of temperature on the hemolytic process. At 4 °C, poor binding of the tolaasin molecules to the erythrocyte membrane was observed and most of the tolaasin molecules stayed in the solution. However, once tolaasin bound to erythrocytes at 37 °C and the temperature was decreased, complete hemolysis was observed even at 4 °C. These results indicate that tolaasin binding to cell membrane is temperature‐sensitive while tolaasin‐induced membrane disruption is less sensitive to temperature change. The effect of erythrocyte concentration was measured to understand the membrane binding and pore‐forming properties of tolaasin. The percentage of hemolysis measured by both hemoglobin release and cell lysis decreased as erythrocyte concentration increased in the presence of a fixed amount of tolaasin. The result shows that hemolysis is dependent on the amount of tolaasin and multiple binding of tolaasin is required for the hemolysis of a single cell. In analysis of dose‐dependence, the hemolysis was proportional to the tenth power of the amount of tolaasin, implying that tolaasin‐induced hemolysis can be explained by a multi‐hit model. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Hemolytic activity of Serratia marcescens

A cell-bound hemolytic activity was found in several strains of Serratia marcescens. One Serratia cell per ten erythrocytes was sufficient to cause complete lysis of human erythrocytes within 2 h in the liquid assay. The hemolytic activity resided in the membrane fraction and could be inactivated by incubating cells with proteases. The hemolytic activity was greatly enhanced in actively metabolizing Serratia cells and was partially controlled by the iron supply. Hemolysis was accompanied by degradation of erythrocyte membrane proteins (band 3 and 6, glycophorin) and was independent of the blood group. The exoprotease secreted by S. marcescens in large amounts was not involved in hemolysis. Comparison with various hemolytic strains of Escherichia coli showed that hemolysis of erythrocytes was more pronounced with S. marcescens than with E. coli. In contrast to hemolysis by E. coli, lysis of erythrocytes by S. marcescens was not enhanced by Ca²⁺ ions.Dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday     

Dark and photosensitized hemolysis of erythrocytes in the presence of dithranol]

Ditranol (1,8-dihydroxy-9-antrone) induced dark lysis of erythrocytes. After irradiation of the cells with UV-light (366 nm UV-A light) in the presence of ditranol (DUVA-effect) the hemolytic effect increases. It has been found that antioxidant butylated hydroxytoluene BHT in the concentration 10(-7) M did not affect the dark lysis, while with increased BHT concentration up to 10(-5) M the hemolytic effect of ditranol was intensified. The presence of BHT in the above concentration under DUVA-effect did not change the velocity of cell lysis. Sodium aside did not affect the dark hemolysis of ditranol, but it inhibited photosensitized hemolysis.     

The relationship of membrane lipids to species specific hemolysis by hemolytic factors from Stomoxys calcitrans (L.) (Diptera: Muscidae)

Posterior-midgut homogenate from female stable flies prepared at 12 h after feeding hemolyzed erythrocytes from 6 different mammalian species more readily than homogenate prepared at 22 h. A significant correlation was obtained between the per cent sphingomyelin content of the erythrocyte membrane and the time required for lysis by the 12 h homogenate. Erythrocytes with low sphingomyelin content were more sensitive to lysis than cells with high sphingomyelin. No such correlation exists for hemolysis by 22 h homogenate. Mean corpuscular volume and osmotic fragilities of erythrocytes were not related to hemolysis either by 12 or 22 h homogenate. Determination of phospholipase C and sphingomyelinase activities showed that the hydrolysis rate of phospholipase C in homogenates prepared at 12–14 h was almost twice as much as sphingomyelinase activity. Whereas hydrolysis rates in 22–24 h homogenate were not different and markedly reduced compared to the 12–14 h homogenate. The times required for erythrocyte hemolysis related to the phospholipase C and sphingomyelinase activity profiles suggests that these enzyme activities participate in the in vitro hemolysis of red blood cells. Bovine and human erythrocytes change their biconcave contour into a spiculated spherical shape when they are exposed to midgut homogenate. This shape change is interpreted as a detergent induced modification of the red cell membrane which renders the erythrocytes more vulnerable to hemolysis.     

Effects of temperature and bovine serum albumin on lysis of erythrocytes induced by dilauroylglycerophosphocholine and didecanoylglycerophosphocholine.

The effects of the incubation temperature and bovine serum albumin on hemolysis induced by short-chain phosphatidylcholine were examined. The rate of hemolysis of human, monkey, rabbit, and rat erythrocytes by dilauroylglycerophosphocholine showed biphasic temperature-dependence: hemolysis was rapid at 5-10 degrees C and above 40 degrees C, but slow at around 25 degrees C. In contrast, the rate of lysis of cow, calf, sheep, pig, cat, and dog erythrocytes did not show biphasic temperature-dependence, but increased progressively with increase in the incubation temperature. Bovine serum albumin increased the hemolysis of human erythrocytes induced by dilauroylglycerophosphocholine or didecanoylglycerophosphocholine: it shortened the lag time of lysis and reduced the amount of phosphatidylcholine required for lysis. A shift-down of the incubation temperature from 40 to below 10 degrees C also shortened the lag time of lysis of human erythrocytes induced by dilauroylglycerophosphocholine and reduced the amount of phosphatidylcholine required for lysis.     

Adenylate cyclase toxin from Bordetella pertussis. The relationship between induction of cAMP and hemolysis.

Bordetella pertussis produces a calmodulin-activated adenylate cyclase (AC) that exists in several forms. Only one form of AC, of apparent 200 kDa, is a toxin that penetrates eukaryotic cells and generates uncontrolled levels of intracellular cAMP. Recombination studies in transposon Tn5-insertion mutants of B. pertussis and amino acid sequence homology with alpha-hemolysin of Escherichia coli suggested that AC toxin may also have a hemolytic activity. Here, we demonstrate that only the toxic form of B. pertussis AC possesses hemolytic activity. Immunoblotting of membranes from sheep erythrocytes throughout the process of cell lysis detects the presence and accumulation of only the 200-kDa form of B. pertussis AC. cAMP generation induced by AC toxin in sheep erythrocytes is immediate whereas appearance of hemolysis is delayed by about 1 h and requires a higher level of AC toxin activity. Addition of exogenous calmodulin to sheep erythrocyte incubation medium potentiates the hemolytic activity of AC toxin but blocks cAMP generation. Extracellular Ca2+ at mM concentrations is absolutely required for cAMP generation but not for hemolysis. However, binding of AC toxin to sheep erythrocytes in the absence of exogenous Ca2+ followed by reincubation of cells in a toxin-free buffer containing Ca2+ leads to an immediate rise in intracellular cAMP. Human erythrocytes bind AC toxin and generate cAMP but are resistant to lysis. These results show that binding of AC toxin to erythrocytes can cause both cAMP generation and hemolysis or only one of these depending on conditions applied and cell type used.     

Effect of retinol on the hemolysis and lipid peroxidation in vitamin E deficiency

A study on the effect of retinolin vitro on the hemolysis of vitamin E deficient rat red blood cells showed that retinol enhanced the lysis of the E deficient cells as compared to the lysis of normal cells. The lipid peroxidation present during hydrogen peroxide induced lysis of E deficient cells was however markedly inhibited in the presence of retinol without affecting the rate of lysis. In an actively peroxidising system of non-enzymatic lipid peroxidation of rat liver or brain homogenates and of brain lysosomes incubated with human erythrocytes, no lysis was obtained; incorporation of retinol in such systems resulted in lysis but no peroxidation. Hydrogen peroxide generating substances almost completely inhibited the lysis of normal human erythrocytes by retinol, but linoleic acid hydroperoxide and auto-oxidised liver or brain homogenates and ox-brain liposomes increased the lysis. It is concluded that vitamin E deficient erythrocyte hemolysis may be augmented by retinol, an anti-oxidant, having a lytic function without the peroxidation of stromal lipids     

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.     

Resistance of human erythrocytes containing elevated levels of vitamin E to radiation-induced hemolysis

Human erythrocytes were isolated from the blood of healthy donors and then incubated in the presence of suspensions of alpha-tocopherol for 30 min at 37 degrees C. Unabsorbed tocopherol was removed by centrifugation using several washes of isotonic phosphate-buffered saline. Washed erythrocytes were resuspended to 0.05%. Hct and exposed to hemolyzing doses of 60Co gamma radiation, and hemolysis was monitored continuously by light scattering at 700 nm in a recording spectrophotometer. The extent of hemolysis with time was sigmoid and data analysis was carried out on the time taken for 50% hemolysis to occur (t50%). The vitamin E content of erythrocytes was significantly elevated by the incubation procedure and resulted in the cells exhibiting a significantly increased resistance to hemolysis as reflected by the extended t50% values. Oral supplementation of 500 IU of vitamin E per day to eight normal human subjects for a period of 16 days also resulted in their washed erythrocytes exhibiting a significant increase in resistance to radiation-induced hemolysis. When comparing vitamin E incubated cells with control cells, both the dose-reducing factor (DRF) and the time for 50% hemolysis quotient (Qt50%) were observed to increase with increasing radiation dose.     

THE EFFECT OF HEMOLYTIC SUBSTANCES ON WHITE CELL RESPIRATION

Substances such as saponin, the bile salts, etc., which produce lysis of red cells also produce cytolysis of white cells from rabbit peritoneal exudates, the arbitrary criterion of their cytolytic effect being their ability to depress the O₂ consumption of the leucocytes. The amount of cytolysis increases regularly as the amount of the added lysin is increased, and sufficiently large quantities of saponin, sodium taurocholate, sodium glycocholate, or sodium oleate are capable of virtually abolishing the O₂ consumption altogether. At the same time, it can be shown that a lysin such as saponin is used up in combining with the white cells in much the same way as it is used up in combining with red cells, and the reduction in oxygen consumption appears to be roughly proportional to the amount so combined. The action of these lytic substances on white cells, in fact, is very similar to their action on red cells, due allowance being made for the fact that the cytolysis of the white cell is probably not an all-or-none process like hemolysis. White cell respiration is also depressed in hypotonic solutions, the respiration being virtually linear with the tonicity.     

Interaction of thermostable direct hemolysin of Vibrio parahaemolyticus with human erythrocytes.

The interaction between thermostable direct hemolysin produced by Vibrio parahaemolyticus WP-1 and human erythrocytes was studied. The lysis of human erythrocytes by the hemolysin was dependent of temperature and no hemolysis occurred at low temperature (0-4 C), but the hemolysin was adsorbed on human erythrocytes even at low temperature. No hemolysis was observed when antihemolysin antiserum was mixed with the hemolysin and human erythrocytes at zero time. On the other hand, lysis of the cells by hemolysin was not completely inhibited when the antiserum was added during the lag time and the inhibitory effect decreased with delay in the time of addition of antiserum. The inhibitory effect of the antiserum decreased with increase in the incubation temperature, increase in the concentration of divalent cations, and decrease in pH. These results suggest that lysis of human erythrocytes by the hemolysin is at least a two-step process consisting of adsorption of the hemolysin to human erythrocytes and the step(s) following adsorption.     

Influence of Glucose Concentration on the Membrane Stability of Human Erythrocytes

The action of glucose as an osmolyte in relation to blood cells is not well-characterized in the literature. This study aimed to study the influence of glucose concentration on the stability of red blood cells. The stability of erythrocytes was evaluated by the half-transition point obtained from the curves of lysis induced by glucose in the absence of salt or by increase in medium hypotonicity in the absence and the presence of different concentrations of glucose. In the presence of 0.9 g/dl NaCl, there was no hemolysis with increasing concentration of glucose from 0 to 10 g/dl. In the absence of NaCl, the dependence of hemolysis with the 0–10 g/dl glucose was described by a decreasing sigmoid, with fully lysed and fully protected cells being encountered in the presence of 0–2 and 4–10 g/dl glucose, respectively. The possible origin of such stabilization effect is discussed with base of what is known about osmostabilization of biological complexes and about the influence of glucose on the rheological properties of erythrocytes.     

alpha-Hemolysin of E. coli]

The activity of alpha-hemolysin increased at the log growth phase in the culture of E. coli P678 Hly+ hemolytic strain; this activity diminished with the change into the stationary phase, and then fell sharply. Replacement of the culture medium in the stationary growth phase by fresh one led to restoration of the hemolytic activity of the culture. The culture fluid separated from the cells at the stationary growth phase produced an inhibitory action on the alpha-hemolysin Ca ions activated and stabilized the alpha-hemolysin. Sodium citrate and sucrose served as hemolysis inhibitors. The action of alpha-hemolysin was maximal against human erythrocytes at pH 6.5. Hemolytic activity was characterized in time by a distinct lag-phase and the phase of the greatest rate of reaction. The duration of the lag-phase and also the rate of hemolysis depended on the concentration of alpha-hemolysin (with the increase of the hemolysin concentration lag-phase was shortened and the reaction was accelerated). There proved to be a linear relationship between the amount of erythrocytes taken into the reaction and the rate of hemoglobin release, and also there was noted a temperature activation of the hemolytic reaction.     

Staphylococcal virolysin,a phage-induced lysin; its differentiation from the autolysis of normal cells

Virolysin is a lysin which appears in Staphylococcus aureus K₁ cells infected with phage P₁₄; together with phage, virolysin is released from phage-infected cells at the time of lysis. Autolysin is a lysin formed by uninfected cells of the K₁ strain; autolysin is released from uninfected cells by autolysis. They show the following similarities: Both agents act within the genus Micrococcus. They lyse cells only after the cell has been subjected to a damaging or "sensitizing" treatment, such as heat, bacteriophage, acetone, or ultraviolet irradiation. The course of lysis of heated cells by both lysins has been found to proceed in a similar manner. A constant percentage of cells is lysed, independent of the concentration of lysin; the residual cells remain resistant to either lysin. Lysis proceeds logarithmically with time, and the velocity constants K are proportional to the lysin concentration. K increases with increasing temperature. Both lysins are unaffected by antiserum to the phage. They are inhibited alike by a number of chemicals, including known enzyme inhibitors. Both agents are destroyed by proteolytic enzymes and are precipitated by 40 per cent saturation with (NH₄)₂SO₄. Both lysins are very thermolabile. The two lysins differ with respect to their pH optimum, antigenic relationship and specificity for Micrococcus lysodeikticus. These results suggest that (1) both lysins have many properties associated with enzymes, (2) the lysis of heated cells, which they produce, has some of the characteristics of a chemical reaction, (3) the lysin from the phage-infected cell is clearly different from the lysin of the uninfected cell.