Hemolysis Of Rabbit Erythrocytes Research Articles

Isolation and characterisation of photoactive haemolytic toxin from Heterocapsa circularisquama

Red tides of Heterocapsa circularisquama, identified as a new species of dinoflagellate a few years ago, have frequently caused mass mortality of several species of bivalve in Japan, whereas no harmful effects on fish have been reported so far. We found that cell-free methanol extract from H. circularisquama caused haemolysis of rabbit erythrocytes and showed cytotoxic effects on HeLa cells and on the rotifer Brachionus plicatilis in a dose- and time-dependent manner. Interestingly, the haemolytic activity and cytotoxic effects of the extract were completely dependent on the presence of light, and no such bioactivities were observed in the dark even at very high concentrations of the extract. The maximum activity was attained at 420nm. These results suggest that H. circularisquama has a photosensitising haemolytic toxin. Highly purified toxin was obtained after several purification procedures including gel-filtration, thin-layer chromatography and reverse-phase high-performance liquid chromatography. The absorption spectrum of this purified toxin in dimethyl sulfoxide showed peaks at 450nm, 590nm and 640nm.

HAEMOLYTIC ACTION OF ALKYLMERCURIC COMPOUNDS AND THEIR INTERACTION WITH A PHOSPHOLIPID MONOLAYER

Haemolysis of rabbit erythrocytes caused by alkylmercuric compounds in vitro and their interaction with a phospholipid monolayer were investigated. Intensity of their haemolytic action was in the following order : n-buty1- > n-propy1- > ethy1-> methyl-mercuric chloride. The interaction of these alkylmercuric chlorides with the phospho-lipid monolayer was the most intensive with n-butyl derivative being followed by other derivatives with the same order as seen in the haemolytic action.In the case of ethylmercuric phosphates, intensity of their interaction with thephospholipid monolayer was also found to be in the same order which has been obtain-ed for intensity of their haemolytic action, namely the order was as follows : tri-> di- > monoethylmercuric phosphate.From these results, a role of the alkyl groups of these compounds to their penetra-tion into erythrocyte membrane was discussed.

Hemolysis of rabbit erythrocytes by Gymnodinium breve toxin.

Hemolysis of rabbit erythrocytes by Gymnodinium breve toxin.

Haemolysis induced by subcellular fractions of rabbit spleen and liver.

The ability of subcellular fractions of rabbit spleen and liver to induce haemolysis of rabbit erythrocytes was investigated. It was shown that: Subcellular fractions of normal spleen and liver induce haemolysis of rabbit erythrocytes modified in vivo by injections of small doses of phenylhydrazine. The haemolytic activity of both tissue preparations was located mainly in the mitochondria and was much higher in the spleen than in the liver. Fresh preparations of the spleen or the liver do not induce haemolysis of intact erythrocytes. When the tissue fractions and also the erythrocytes were obtained from phenyl‐hydrazine treated rabbits, the haemolytic activity of the spleen preparations was lower and that of the liver was slightly higher than the respective activities of preparations of normal tissues. The haemolytic activity of the subcellular fractions kept frozen for 7 days at pH = 7.0 was significantly increased. Phospholipase A is indicated as a possible haemolytic agent present in the spleen and the liver mitochondria.

Effect of pH on the haemolysis of rabbit erythrocytes by staphylococcus alpha--toxin.

Summary The effect of toxin concentration on the kinetics of haemolysis of rabbit erythrocytes has been studied at pH values differing by about 0·5 pH units over the range 5·5 to 8·5 for both crude and partially purified toxin at concentrations of 2·5, 5·0, 10, 20, 30, 40, 60 MHD per ml and at 80 and 120 MHD per ml for the partially purified toxin. Increased concentration of toxin caused a decrease in the prelytic lag time (·) of the sigmoid haemolysis curves, a decrease in the time to reach 50 per cent. haemolysis (t1/2), and an increase in the maximum rate of haemolysis (Rm). Plateaux in Rm-toxin concentration, ·-toxin concentration and t1/2-toxin concentration curves were found at all pH values: these were displaced to higher concentrations in plots drawn for the partially purified toxin. The effect of pH on haemolysis is very marked, and pH profiles for the maximum rate of haemolysis were constructed: for the crude toxin, the shape of the profile changed as the toxin concentration was increased, while for the partially purified toxin the shape was independent of toxin concentration. The pattern of the profiles indicates that the crude toxin contains a small concentration of impurity that is strongly lytic at pH 5·5 and less markedly so at 8·5; the impurity has no significant effect at intermediate pH values. The reaction is first order with respect to toxin over a very small range of concentrations only, and the maximum rate of haemolysis is independent of toxin concentration at concentrations greater than 120 MHD per ml. The reaction is first order with respect to erythrocytes at all pH values, and over all measurable concentrations.

The hydrational effect of leptazol and its theoretical connection with glucose deficiency in the haemolysis of rabbit erythrocytes

1. 1. The activation parameters for the malonamide-induced haemolysis of rabbit erythrocytes in the presence of leptazol indicate that this drug is an hydration-structure breaker, isokinetic with malonamide and, theoretically, isokinetic with glucose. 2. 2. At concentrations near to those which induce convulsions, the activation parameters for leptazol and glucose are close together, indicating comparable effects on erythrocyte hydration structure. 3. 3. These observations are discussed, and it is suggested that the convulsions induced by leptazol, insulin hypoglycaemia and electric-shock treatment may depend on the disruption of cerebral hydration structure.

Rabbit erythrocyte hemolysis by lipophilic, aryl molecules.

SummaryIndole, skatole, and 17 additional aryl molecules can interact with the cellular membrane to produce hemolysis of the rabbit erythrocyte. The minimum concentrations of aryl molecules required to produce total lysis of 1.4 × 108 red blood cells are correlated with the lipophilicity of the molecules as measured by their partitioning across the interface barrier of n-octanol: aqueous phosphate buffer, 50 mM, pH 7.4, phases. Hemolysis requires lesser amounts of a molecule that is lipophilic than one that is hydrophilic.

The role of hydration in the effect of barbiturates on the haemolysis of rabbit erythrocytes

1. 1. An evaluation has been made of the effect of barbiturates on the temperature dependence of haemolysis of rabbit erythrocytes. 2. 2. It has been found that the kinetics conform with the compensation law, and this shows that hydrational effects predominate in the mechanism of the process. 3. 3. The results indicate that the alkyl groups of barbiturates support apolar hydration, and the suggestion is made that this may be connected with pharmacological activity.

Reevaluation of heterophile hemolytic response in psychiatric patients.

IN 1947, Sjovall¹reported occurrence of hemoglobin in the urine of mice following injection of serum of schizophrenics and loss of this activity of serum following heating at 56 C for one half hour. Turner and Chipps pursued this lead and in 1966 reported²that there is an antirabbit heterophil hemolysin of widespread occurrence in human serum. Their study indicated that this hemolysin was present at higher levels in the serum of schizophrenics than those of other persons. The interest aroused by this notion of heterophil hemolysins as another³potential "biochemical lesion" in schizophrenia prompted further investigation. Operating essentially within the same framework as Turner and Chipps, the data we collected fails to indicate a difference between schizophrenic and nonschizophrenic populations with respect to serum hemolysis of rabbit erythrocytes. <h3>Methods</h3> Blood was drawn in the morning from fasting subjects. One part of the

Studies on the homogeneity of prymnesin, the toxin isolated from Prymnesium parvum Carter

Prymnesin, the purified toxin of Prymnesium parvum Carter, was subjected to a variety of inactivation procedures: Irradiation by visible or ultraviolet light, incubation at 37° and exposure to alkaline media. Inactivation was measured by three assay methods: Lethality for fish, hemolysis of rabbit erythrocytes, and inhibition of the acetylcholine-induced contractions of the small intestine of the guinea pig. It was observed that the rate of destruction of the antispasmodic activity exceeds by far the rate of decline of the ichthyotoxic or hemolytic potency. The heterogeneity of prymnesin is thus established.

HEMOLYSIS OF RABBIT ERYTHROCYTES BY PURIFIED STAPHYLOCOCCAL ALPHA-TOXIN. II. EFFECTS OF INHIBITORS ON THE HEMOLYTIC SEQUENCE.

Cooper, Louis Z. (New England Center Hospital, Boston, Mass.), Morton A. Madoff, and Louis Weinstein. Hemolysis of rabbit erythrocytes by purified staphylococcal alpha-toxin. II. Effect of inhibitors on the hemolytic sequence. J. Bacteriol. 87:136-144. 1964.-Study of the time course of hemolysis of rabbit erythrocytes by purified staphylococcal alpha-lysin revealed that the specific toxin-red cell reaction occurs during the prelytic period. This reaction could be prevented or decreased by alpha-lysin antitoxin added early, but not by antitoxin added at the end of the prelytic phase or at any time thereafter. In contrast, hemolysis is suppressed temporarily by sucrose and permanently by polyethylene glycol, even when these are added during the period of rapid release of hemoglobin. When sucrose is present together with alpha-lysin and red cells only during the prelytic period, and when the cells are then washed and resuspended in phosphate-buffered saline, their subsequent hemolysis is not altered by the presence of the sugar. This is not so when antitoxin is employed. When erythrocytes are laked by a measured excess of alpha-lysin, only a portion of the original hemolytic activity can be recovered. Repeated exposure of lysin to red cells produces a loss of activity represented by a linear function when logs of residual activity are plotted sequentially. Once alpha-lysin has reacted with red cells, it does not appear to be available for attachment to other erythrocytes.

HEMOLYSIS OF RABBIT ERYTHROCYTES BY PURIFIED STAPHYLOCOCCAL ALPHA-TOXIN I

Cooper, Louis Z. (New England Center Hospital, Boston, Mass.), Morton A. Madoff, and Louis Weinstein. Hemolysis of rabbit erythrocytes by purified staphylococcal alpha-toxin: I. Kinetics of the lytic reaction. J. Bacteriol. 87:127-135. 1964.-The hemolytic activity of purified staphylococcal alpha-lysin was found to be directly proportional to toxin concentration and inversely related to the log concentration of rabbit erythrocytes. Activity was directly proportional to the duration of lysin-red cell incubation until inactivating effects of heat and dilution became significant; this linear relationship was prolonged by incubation at a lower temperature and addition of bovine serum albumin. Study of the time course of hemolysis at different alpha-lysin concentrations revealed a family of sigmoid curves characterized by a prelytic lag phase and a period of rapid linear release of hemoglobin. The duration of prelytic lag varied inversely with the quantity of toxin, but the rate of hemolysis was directly proportional to toxin and red-cell concentrations. The presence of bovine serum albumin decreased the prelytic lag, prolonged the linear phase of the reaction, and increased total hemolysis. In the range of 25 to 46 C, the prelytic lag period became shorter with increase in temperature; at 48 to 52 C, it was markedly prolonged and hemolysis was strikingly diminished. As the incubation temperature was increased from 25 to 52 C, there was a decrease in the degree of maximal hemolysis, presumably due to thermal inactivation of alphalysin. The rate of hemolysis, when measured to 50% hemolysis, was optimal between 34 and 42 C but, when determined to the 10% level, was greatest between 40 and 46 C. The features of the hemolytic reaction suggest that staphylococcal alpha-toxin has the characteristics of an enzyme.

HEMOLYSIS OF RABBIT ERYTHROCYTES BY PURIFIED STAPHYLOCOCCAL ALPHA-TOXIN. III. POTASSIUM RELEASE.

Madoff, Morton A. (New England Center Hospital, Boston, Mass.), Louis Z. Cooper, and Louis Weinstein. Hemolysis of rabbit erythrocytes by purified staphylococcal alpha-toxin. III. Potassium release. J. Bacteriol. 87:145-149. 1964.-The reaction between staphylococcal alpha-toxin and erythrocytes was characterized by rapid release of K(+) from the cells, early in the prelytic period; 50 to 75% of this loss occurred before leakage of hemoglobin was detectable. The addition of specific antitoxin early enough in the reaction to inhibit gross hemolysis also inhibited cation release. The presence of sucrose or polyethylene glycol prevented hemoglobin release, but was without effect on K(+) leak. These observations suggest that K(+) loss is a more specific indication of the progress of the reaction between alpha-toxin and erythrocytes than is the release of hemoglobin.

Behavior of erythrocytes in various solvent systems I. Water‐glycerin and water‐propylene glycol

The hemolytic behavior of rabbit and human erythrocytes in water‐glycerin and water‐propylene glycol solutions was investigated. Complete hemolysis of erythrocytes took place in all propylene glycol solutions and most glycerin solutions. Aqueous solutions containing 50, 60, and 70 per cent glycerin prevented complete hemolysis of rabbit erythrocytes but not human erythrocytes. The addition of sodium chloride to various glycerin solutions prevented hemolysis. The addition of sodium chloride to propylene glycol solutions prevented hemolysis of rabbit erythrocytes in 5–30 per cent solutions and of human erythrocytes in 5–40 per cent solutions. When possible, the data were used to calculate van't Hoff i values for sodium chloride in various water‐glycerin and water‐propylene glycol solutions. Unusual behavior was displayed by erythrocytes in 40–50 per cent propylene glycol solutions. The addition of sodium chloride to solutions containing 50 per cent or more of propylene glycol did not prevent complete laking of red blood cells.