Shape and Volume Changes in Frog Erythrocytes Following Ultraviolet Radiation

Frog erythrocytes in Ringer's solution were exposed to ultraviolet radiation and then followed in camera lucida drawings for changes in shape and dimension. Cell thickness was found to increase while cell width remained constant throughout the period prior to hemolysis. The cell shortened and bulged at the ends during the middle third of the prolytic period while a region around the cell center remained constricted. When this constricted region gave way, the cell became spherical and hemolyzed. Cell volume as calculated from the cell's dimensions increased linearly with time throughout the prolytic period to hemolysis then dropped rapidly to a constant value somewhat higher than the original cell volume. These changes in shape and volume are consistent with a colloid osmotic type of hemolysis but with other factors acting to limit the rate of swelling and the forms assumed during the swelling process. The relationship between the time of hemolysis and the cell surface area exposed to the ultraviolet is discussed as it applies to the site of ultraviolet damage.     

PROLYTIC ION EXCHANGES PRODUCED IN HUMAN RED CELLS BY METHANOL,ETHANOL, GUAIACOL,AND RESORCINOL

When the washed red cells of heparinized human blood are exposed at 4°C. to methanol, ethanol, guaiacol, or resorcinol in hypolytic concentrations in isotonic NaCl, the prolytic loss of K at the end of 20 hours varies from about 25 per cent of the initial K content of the cells in the case of 3.1 M methanol to about 55 per cent of the initial K in the case of 0.04 M resorcinol. As in the case of the prolytic losses observed with other lysins, the K loss is rapid at first and then slows down so that what appears to be a new steady state is reached logarithmically. The K lost from the cells during the period of the prolytic loss is replaced by an approximately equivalent amount of Na, derived from the isotonic NaCl in which the cells are suspended. The Na which enters can be replaced by K by washing the cells in isotonic KCl, and this K can again be replaced by Na by washing the cells in isotonic NaCl. The remainder of the cell K., i.e. the K which was not lost during the period of the prolytic loss, is retained in the cell unaffected by these washing procedures. The capacity of red cells for undergoing disk-sphere transformations is scarcely affected by their having been exposed to hypolytic concentrations of methanol, ethanol, guaiacol, or resorcinol in isotonic NaCl, and their resistance to osmotic hemolysis and to lysis by saponin and digitonin is altered only in minor respects even when as much as 50 per cent of the cell K has been exchanged for Na. Some restriction to the movement of K between the cell and its environment is apparently modified irreversibly when the cell is exposed to hypolytic concentrations of lysins, and the modification is such that only a fraction of the cell K is affected, the fraction being a function of the lysin concentration, the duration of its action, and other factors. A modification of some part of the cell structure and of the properties dependent on its integrity is probably involved: K may be lost more readily from some cells than from others, from some parts of the cell more readily than from other parts, or the explanation may lie in changes in the extent to which Hb binds ions or in modifications of metabolic processes.     

THE PROLYTIC LOSS OF K FROM HUMAN RED CELLS

The prolytic loss of K., i.e. the loss of K which takes place from red cells exposed to hypolytic concentrations of lysins, has been measured in systems containing distearyl lecithin, sodium taurocholate, sodium tetradecyl sulfate, saponin, and digitonin, by means of the flame photometer. The lysins are added in various concentrations to washed red cells from heparinized human blood, and the K in the supernatant fluids is determined after various intervals of time and at various temperatures. The prolytic loss K_p is compared in every experiment with the loss K_s into standard systems containing isotonic NaCl alone, with no lysin. The losses K_s and K_p increase with time, so that new steady states are approached logarithmically. The values of K_p which correspond to the new steady states depend on the lysin used, being greatest with taurocholate and smallest with digitonin. The temperature coefficient of the loss is positive, and the extent and course of the losses have no apparent relation to the prolytic shape changes. In systems in which the loss of K is appreciable, it can be inhibited by the addition of plasma or of either cholesterol or serum albumin. Of these two substances, even when used in quantities which have an approximately equal effect in inhibiting hemolysis, serum albumin is much the more effective. Just as the prolytic loss of K occurs without the loss of any Hb, so in concentrations of lysin sufficient to produce hemolysis the loss of K, expressed as a percentage of the total red cell K, increases much more rapidly with lysin concentration than does the loss of Hb expressed as a percentage of the total Hb. The explanation of these relations depends on whether the loss of K is treated as being all-or-none in the case of the individual cell or as being the result of the loss of part of the K from all of the cells. This point has still to be decided.     

Photodynamic hemolysis at low temperatures

It is shown that photodynamic hemolysis may occur at –79°C. if the erythrocytes are suspended in a solution containing 70 per cent glycerol which prevents hemolysis by freezing; but that there is no hemolysis under the same conditions at –210°C. At the higher temperature the viscosity of the solution is still low enough to permit appreciable movement of molecules, whereas at the lower temperature the molecules must be virtually immobile. The findings are compatible with the idea that the dye molecule acts in a cycle, bringing about successive oxidations by O₂ molecules, as has been shown for photodynamic hemolysis at room temperature. The assumption of a combination between dye, O₂, and substrate does not explain photosensitized hemolysis in the semi-solid state. The mechanism of photosensitized oxidation by O₂ is discussed.     

Particularities in the kinetics of growth and citric-acid accumulation by saccharomycoposis lipolytica

When glucose is used as C-source extracelluar citric-acid accumulation does not start at the point of N-source exhaustion. A transition phase lasting for about 2–3 hours is observed. It may be assumed that the continuation of growth after the N-source exhaustion coincides with the delay of the citric-acid accumulation. During the course of citric-acid production characteristic interruptions of the glucose uptake and the citric-acid accumulation are found. The findings are discussed in more detail.     

Time-dependent Mechanisms in Beta-cell Glucose Sensing

The relation between plasma glucose and insulin release from pancreatic beta-cells is not stationary in the sense that a given glucose concentration leads to a specific rate of insulin secretion. A number of time-dependent mechanisms appear to exist that modify insulin release both on a short and a longer time scale. Typically, two phases are described. The first phase, lasting up to 10 min, is a pulse of insulin release in response to fast changes in glucose concentration. The second phase is a more steady increase of insulin release over minutes to hours, if the elevated glucose concentration is sustained. The paper describes the glucose sensing mechanism via the complex dynamics of the key enzyme glucokinase, which controls the first step in glucose metabolism: phosphorylation of glucose to glucose-6-phosphate. Three time-dependent phenomena (mechanisms) are described. The fastest, corresponding to the first phase, is a delayed negative feedback regulating the glucokinase activity. Due to the delay, a rapid glucose increase will cause a burst of activity in the glucose sensing system, before the glucokinase is down-regulated. The second mechanism corresponds to the translocation of glucokinase from an inactive to an active form. As the translocation is controlled by the product(s) of the glucokinase reaction rather than by the substrate glucose, this mechanism gives a positive, but saturable, feedback. Finally, the release of the insulin granules is assumed to be enhanced by previous glucose exposure, giving a so-called glucose memory to the beta-cells. The effect depends on the insulin release of the cells, and this mechanism constitutes a second positive, saturable feedback system. Taken together, the three phenomena describe most of the glucose sensing behaviour of the beta-cells. The results indicate that the insulin release is not a precise function of the plasma glucose concentration. It rather looks as if the beta-cells just increase the insulin production, until the plasma glucose has returned to normal. This type of integral control has the advantage that the precise glucose sensitivity of the beta-cells is not important for normal glucose homeostasis.     

Factors affecting the hemolytic action of "lysolecithin" upon rabbit erythrocytes

1. Lysolipid was prepared by the action of snake venom on egg yolk, and a study was made of the factors affecting its hemolytic action upon rabbit erythrocytes. 2. Lysis proceeded very rapidly at first, then ceased within a few minutes at room temperature. A given amount of lysin appeared to hemolyze a fixed number of cells, under specified conditions. 3. The more dilute erythrocyte suspensions required relatively more lysin per cell, for 50 per cent hemolysis of the suspension. There may be an equilibrium between the lysin dissolved in the medium and that adsorbed on the cells. 4. The degree of hemolysis for varying lysin concentrations was measured, and the cells showed a typical distribution of resistance to hemolysis. 5. As the temperature was lowered lysis was more extensive. Adsorption of the lysin on the cell surface was apparently increased. 6. The resistance of the erythrocytes to lysis increased slightly as the pH was raised from 5.5 to 7.8. 7. Resistance to lysis was independent of the tonicity of the medium and of initial cell volume. The magnitude of the cell surface was probably the determining factor. 8. A marked shrinkage of the erythrocytes was observed in the presence of calcium ions and lysin, but not in the absence of the lysin. 9. Hemolytic resistance curves obtained by the Wilbrandt technique were of the "colloid-osmotic" type. However, there was no evidence of prolytic loss of potassium ions. 10. Hypotonic fragility of the cells was slightly increased in the presence of the lysin. The rate of penetration of thiourea was greatly increased.     

Biosynthesis of Thiazole in Exobasidium

In some Exohmidium strains, which grow on a synthetic medium containing glucose citralc, and thiamine as the only organic compounds, growth usually does not start until after more than 800 honrs if thiamine is replaced by pyrimidine. Homocysteine and methionine are able to shorten this lag phase of E. vaccinil“myrt.” Methionine is growth-promoting only at about pH 4. At low concentrations the yield is proportional to the concentration of methionine. Growth ceases when the methionine is consumed and the fungus then enters a lag phase as long as that in methionine-free medium. Methionine does not indnce the thiazole-synthesizing enzymes, but is probably consumed as a precursor of thiazole. The utilization of this precursor is delayed by some growth factors and amino acids, i.e. cholin, betaine, glutamic acid or glutamine. The delayed adaptation of the methionine- and thiazole-synthesizing enzymes seems to be regulated by changes in the nutritional state of the ageing cultures. The mechanism of this regulation has not been settled.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing sucrose: relationship with posthypertonic hemolysis and solute movements

Human red blood cells were frozen at temperatures down to ?9 °C in solutions containing sucrose, and the hemolysis on thawing was measured. This was compared with the hemolysis caused by exposing the cells to high concentrations of sucrose and then resuspending them in more dilute solutions at 4 °C. The effects of the hypertonic solutions of sucrose on potassium, sodium, and sucrose movements were also investigated. It was found that sucrose does not prevent damage to the cells by very hypertonic solutions (whether during freezing and thawing or at 4 °C) but it does reduce hemolysis of cells previously exposed to these solutions if present in the resuspension (or thawing) solution. Evidence is presented that the damaging effects of the hypertonic solutions of sucrose occurring during freezing are associated with changes in cell membrane permeability but that posthypertonic hemolysis is not primarily associated with a “loading” of the cells with extracellular solutes in the hypertonic phase. It is concluded that sucrose may reduce hemolysis of red blood cells by slow freezing and thawing by reducing colloid osmotic swelling of cells with abnormally permeable membranes.     

The mechanism of the hemolytic activity of polyene antibiotics

The kinetics of the filipin-, amphotericin B- and nystatin-induced hemolysis of human erythrocytes were investigated. Filipin-induced hemolysis is of the damage type. It is an all-or-none process, partly inhibited by Ca2+ or Ba2+ but not by Mg2+, Na+ or SO42-. The hemolytic activity of filipin is explained by the formation of large aggregates within the erythrocyte membrane in the form of large perforations, permeable to substances of low molecular weight as well as to macromolecules, including hemoglobin. In isotonic KCl solution, both amphotericin B and nystatin, at low concentrations, form smaller aggregates within the membranes. As a result, the permeability of the membranes to KCl increases and hemolysis occurs. However, the kinetics of the hemolysis induced by the two polyenes is complex. The process shows some features of the permeability type and some of the damage type. It is suggested that amphotericin B and nystatin may simultaneously form a number of transport systems, differing in their molecular organisation and hemolytic activity. Their participation in erythrocyte membrane permeability can be modified by small changes in membrane organisation and the chemical composition of the incubation medium. In isotonic solutions of divalent cation chlorides, and at higher antibiotic concentration, additional aggregates, allowing divalent cations to permeate, appear. These structures do not permit SO4(2-) to permeate.     

The mechanism of bile salt-induced hemolysis.

The hemolytic activities of sodium deoxycholate (DChol) and its tauro-conjugate (TDChol) and glyco-conjugate (GDChol) were analysed. 50 % hemolysis occurred in 30 min at pH 7.3, at the concentrations of these detergents equal to 0.044, 0.042 and 0.040 % respectively. These values are below their critical micellar concentrations. Based on its kinetics, this hemolysis is classified as being of permeability type. The detergents increase the permeability of erythrocyte membranes to KCl, and colloid osmotic hemolysis occurs. The minimum of hemolytic activity of the three cholates is at about pH 7.5. A very high increase in hemolytic activity occurs at pHs below 6.8, 6.5 and 6.2 for DChol, TDChol, and GDChol, respectively. These values are close to the pK(a) for DChol (6.2), but much higher than the pK(a) for TDChol (1.9) and GDChol (4.8). It is therefore suggested that the increase in hemolytic activity is not a result of the protonation of the anionic groups of the cholates. At acidification below pH 6, the kinetics of DChol induced hemolysis change to the damage type characterised by nonselective membrane permeability. Such a transition is not observed in TDChol and GDChol induced hemolysis. It is therefore suggested that the change in the type of hemolysis depends on protonation of the anionic group of cholates.     

Regulation of glycolysis in the mouse blastocyst during delayed implantation

The rate of oxidation of glucose is reduced in mouse embryos in the prolonged free living phase associated with delayed implantation and increases when the embryos are reactivated by estrogen. To determine how these changes in metabolism are regulated, several aspects of glucose metabolism were evaluated in dormant and reactivated blastocysts: 1) Embryos were exposed to 14C-pyruvate in vitro and evolved 14CO2 was measured. It was found that the rate of production of CO2 was equal in the two types of blastocysts, suggesting that aerobic pathways are fully functional during delayed implantation. 2) Production of lactate in the presence of O2 was measured and a decrease of 30% was found in delayed implanting embryos, suggesting that the overall regulatory mechanism for glucose metabolism resides in the glycolytic portion of the pathway. 3) Capacity for uptake and phosphorylation of glucose was evaluated using 3H-2-deoxyglucose and was found to be equal in the two types of embryos. 4) Total amounts of the rate-controlling enzymes for glycolysis (i.e., hexokinase and phosphofructokinase) in lysates of delayed and reactivated embryos were found to be equal, indicating that amounts of these enzymes are not limiting in delayed implantation. 5) Lactate production, measured under anaerobic conditions, was found to be equal, demonstrating that it is not the capacity for glycolysis but a difference in the degree of allosteric inhibition that is responsible for reduced glucose oxidation in delayed implantation. 6) Levels of ATP, ADP, and hexose-6-phosphates were found to be consistent with allosteric inhibition of the glycolytic pathway at phosphofructokinase during delay and a release of this inhibition with reactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hemolysis of human red blood cells by freezing and thawing in solutions containing polyvinylpyrrolidone: relationship with postthypertonic hemolysis and solute movements

An investigation was made into the effects of the presence of polyvinylpyrrolidone (PVP) on changes in human red blood cells suspended in hypertonic solutions, on posthypertonic hemolysis, and on freezing at temperatures down to ?12 °C.PVP is very effective at reducing hemolysis when the red blood cells are frozen at temperatures down to ?12 °C. However, the membranes of the cells recovered on thawing have become very permeable to sodium and potassium ions and there is a much increased hemolysis if the cells are resuspended in an isotonic solution of sodium chloride.The presence of PVP does not affect the dehydration of the cells or the development of a change in membrane permeability when the cells are shrunken in hypertonic solutions at 0 °C. Neither does its presence in the hypertonic solution reduce the extent of posthypertonic hemolysis at 4 °C (as measured by the hemolysis on resuspension in an isotonic solution of sodium chloride), but it is more effective than sucrose at reducing hemolysis when present in the resuspension solution. It is concluded that the PVP is able to prevent swelling and hemolysis of cells which are very permeable to cations by opposing the colloid osmotic pressure due to the hemoglobin. However, this does not explain how PVP is able to protect cells against freezing damage at high cooling rates, and a mechanism by which it might do this is discussed.     

Investigations into combined dietary deficiencies of copper,selenium, and vitamin E in the rat

Interactions between dietary Cu, Se, and vitamin E in ascorbate-induced hemolysis of erythrocytes obtained from rats fed diets deficient or adequate in these elements were investigated. Hemolysis was affected by all three dietary factors, through closely interrelated but distinct mechanisms. In vitamin E-deficient cells, hemolysis was increased and the amount of hemolysis was directly related to the amount of hemoglobin breakdown. Deficiency of Cu or Se decreased hemolysis, but only in vitamin E-deficient cells. Vitamin E did not affect the breakdown of hemoglobin, but Cu and Se did. Hemolysis and hemoglobin breakdown were decreased by the addition of glucose, through mechanisms independent of that involving reduced glutathione metabolism. These results suggest that vitamin E acts within erythrocyte membranes to prevent products of hemoglobin breakdown from initiating peroxidation and subsequent hemolysis. Effects of Cu and Se are linked with that of vitamin E by the involvement of glutathione peroxidase and Cu superoxide dismutase in the cytoplasmic breakdown of hemoglobin, rather than by a direct effect of these enzymes on lipid peroxidation. It is concluded that the erythrocyte, because of its high heme content, probably represents a special system in terms of peroxidative pathways, and these findings may not be directly applicable to other tissues.     

Comparison of acid and alkaline hemolysis mechanisms in human erythrocytes

A comparative analysis of the mechanisms of base- and acid-induced hemolysis was performed. The results obtained indicate the transport of base equivalents through the anion exchanger during the initial phase of base-induced hemolysis, followed by oxidative stress on cellular membranes and hemolysis. It was shown that the Ellman's reagent (0.4 mM) did not prevent NaOH-induced hemolysis but fully inhibited HCL-induced hemolysis. The inhibition of acid-induced hemolysis was accompanied by the crosslinking membrane proteins, presumably through their acylation. The addition of SH-reducing reagents (cystein, dithiotreitol and, to a lesser extent, albumin eliminated the crosslinkage of membrane proteins and impaired the permeability barrier. It was found that crosslinkage could not prevent the oxidative damage of membrane proteins but was able to preserve the permeability barrier. Based on these results, it was concluded that the barrier impairments associated with acid-induced hemolysis were due to the aggregation of membrane proteins that underwent oxidative damage.     

Relationship between the hemolytic action of heavy metals and lipid peroxidation

It is well known that some of the heavy metals have a hemolytic action, but the mechanisms responsible for this effect are not well established. In order to elucidate whether or not the hemolytic action of heavy metal ions is associated with the peroxidation of membrane lipids, the relationship between metal-induced hemolysis and the generation of malonaldehyde has been studied.The results obtained show that metal-induced hemolysis is associated with the development of peroxidative processes in erythrocyte membranes. The peroxidation is caused by metals with and without pro-oxidant catalytic action. The level of the malonaldehyde products rises before the appearance of hemolysis which proves that the development of peroxidative processes precedes but does not result from hemolysis.The suggestion has been made that the peroxidation of membrane lipids is a possible mechanism of damage to the red cell membrane in metal-induced hemolysis.     

Oligomeric behavior of Bordetella pertussis adenylate cyclase toxin in solution

Adenylate cyclase (AC) toxin from Bordetella pertussis inserts into eukaryotic cells, producing intracellular cAMP, as well as hemolysis and cytotoxicity. Concentration dependence of hemolysis suggests oligomers as the functional unit and inactive deletion mutants permit partial restoration of intoxication and/or hemolysis, when added in pairs [M. Iwaki, A. Ullmann, P. Sebo, Mol. Microbiol. 17 (1995) 1015-1024], suggesting dimerization/oligomerization. Using affinity co-precipitation and fluorescence resonance energy transfer (FRET), we demonstrate specific self-association of AC toxin molecules in solution. Flag-tagged AC toxin mixed with biotinylated-AC toxin, followed by streptavidin beads, yields both forms of the toxin. FRET measurements of toxin, labeled with different fluorophores, demonstrate association in solution, requiring post-translational acylation, but not calcium. AC toxin mixed with DeltaR, an inactive mutant, results in enhancement of hemolysis over that with wild type alone, suggesting that oligomers are functional. Dimers and perhaps higher molecular mass forms of AC toxin occur in solution in a manner that is relevant to toxin action.     

Effect of hexachlorophene on monovalent cation transport in human erythrocytes a mechanism for hexachlorophene-induced hemolysis

Hexachlorophene-induced hemolysis, as studied by phase contrast microscopy, appeared to be a result of osmotic swelling. Both swelling and subsequent hemolysis were markedly delayed by addition of the non-penetrating solute sucrose to the incubation mixture. Binding studies indicated that hexachlorophene is associated primarily with the erythrocyte membrane, the remainder being found in the cytoplasm. Hexachlorophane induced a dose-dependent, first-order efflux of Na⁺ and K⁺ from red cells. The rates of hemolysis and K⁺ efflux induced by hexachlorophene were much greater than would be expected if this compound were acting simply as a metabolic inhibitor and/or an inhibitor of (Na⁺-K⁺-Mg²⁺)-ATPase. It is suggested that hexachlorophene induces the efflux of Na⁺ and K⁺ from red cells by directly altering the permeability of the cellular membrane. Further, hexachlorophene-induced hemolysis is probably a secondary event resulting from osmotic swelling subsequent to increased membrane permeability.     

Mechanism of human erythrocyte hemolysis induced by short-chain phosphatidylcholines and lysophosphatidylcholine

The incorporation and accumulation of a certain amount of short-chain phosphatidylcholine or lysophosphatidylcholine into lipid bilayers of erythrocyte membranes is the first step causing membrane perturbation in the process of hemolysis. Accumulation of dilauroylglycerophosphocholine into membranes makes human erythrocytes "permeable cells"; Ions such as Na+ or K+ can permeate through the membrane, though large molecules such as hemoglobin can not. The "pore" formation was partially reproduced in liposomes prepared from lipids extracted from human erythrocyte membranes; C12:0PC induced the release of glucose from liposomes but did not significantly induce the release of dextran. It was suggested that the phase boundary between dilauroylglycerophosphocholine and the host membrane bilayer or dilauroylglycerophosphocholine rich domain itself behaves as "pores." Erythrocytes could expand to 1.5 times the original cell volume without any appreciable hemolysis when incubated with C12:0PC at 37 degrees C. The capacity of the erythrocytes to expand was temperature dependent. The capacity may play an important role in the resistance of the cells against lysis. The "permeable cell" stage could be hardly observed when erythrocytes were treated with didecanoylglycerophosphocholine and lysophosphatidylcholine. Perturbation induced by accumulation of didecanoylglycerophosphocholine or lysophosphatidylcholine may cause non specific destruction of membranes rather than formation of a kind of "pore."     

Acute immune hemolysis induced by a degradation product of amphotericin B

We report here on an eight-year-old boy who first developed acute intravascular hemolysis following therapy with amphotericin B (AmB) and subsequently a delayed hemolytic transfusion reaction due to alloantibodies. Although there is as yet no evidence for metabolism of AmB in vivo, the hemolysis appeared to be the result of sensitization against a degradation product of the drug. The patient's serum contained a hemagglutinating IgM antibody that reacted with all red blood cells (RBC) tested in the presence of plasma obtained from patients receiving AmB (ex vivo antigen), but not in the presence of their urine, AmB itself, or with AmB-pretreated RBC. These findings indicate that the antibody was directed against a degradation product of AmB, presumably a trace metabolite, that has not yet been identified.