The effects of 3-methylindole on hemolysis, transport of Na+, and ATPase activities of bovine erythrocytes.

Biochemical effects of 3MI on cellular membranes were investigated. This study was conducted to examine the effects of 3MI on the hemolysis of erythrocytes, the transport of 22Na+ in resealed erythrocyte ghosts, and on the ATPase activities of erythrocyte membranes. The percent of hemolysis as a function of 3MI incubation time was sigmoidal. Seventy-five percent of the hemoglobin was released with the second 2 hr of incubation during which the concentration of 3MI in the cells reached a plateau of 2500 mug/ml of packed RBC. The effect of 3MI at a subhemolytic concentration on passive and active 22Na+ transport were not significant. The total and Mg2+-dependent ATPase activities in the membranes were significantly increased after 1 hr of incubation with 3MI at concentrations of 100, 200, 300, 400 and 500 mug/ml (P less than or equal to 0/ml (P smaller than or equal to 0.02).     

Large differences in erythrocyte stability between species reflect different antioxidative defense mechanisms

We have developed a screening assay for erythrocyte stability, which is rapid, easy, inexpensive, robust, and suitable for handling a large number of samples in parallel. Erythrocytes are incubated overnight in 96-well microtiter plates in absence or presence of various oxidants, intact cells are pelleted by centrifugation, and lysis is determined by release of intracellular constituents into the supernatant as either activity of lactate dehydrogenase (LDH) or absorbance of hemoglobin at 406 nm. There is good correlation between the methods. A number of advantages by the present method are that only small amounts of blood is needed, washing is optional, erythrocytes may be stored for at least one day before assay, and large numbers of samples can be handled in parallel. Using this set-up, we have compared erythrocyte stability from several different animal species. We find that erythrocyte susceptibility towards lysis induced by H₂O₂ and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) is highly species dependent. The different susceptibility between species is due to cellular components, since swapping of plasma between species has little or no effect. As a novel observation, we find that erythrocytes from chicken are the most sensitive of the species tested towards lysis by H₂O₂ and are almost four orders of magnitude more sensitive than erythrocytes from man. This is due to a much lower content of catalase in erythrocytes from chicken. A more narrow range is observed for susceptibility towards AAPH and the ranking between the species is different. Thus, chicken erythrocytes are more resistant towards AAPH than some mammals by up to two orders of magnitude. This differential stability towards different oxidative stressors is likely due to evolution/selection of different defense mechanisms.     

Comparative study of the effect of phenazine methosulfate and vitamin K3 on human erythrocytes by hemolysis curves]

Hemolysis curves were used for comparative study of phenazine methylsulfate (PMS) and vitamin K3 action on human erythrocytes. Some differences in PMS and vitamin K3 action were revealed while the concentration of studied compounds and incubation time with them were changed. It is considered that the observed differences in PMS and vitamin K3 action are caused by different degree of oxidation of intracellular hemoglobin.     

Lysosomal breakdown of erythrocytes in the sheep placenta

Summary The breakdown of erythrocytes within the lysosomal apparatus of trophoblastic epithelial cells of the sheep placenta was studied at the ultrastructural level. Acid phosphatase activity could be demonstrated in the interspace between the erythrocyte membrane and the lysosomal membrane, but not inside ingested erythrocytes. The erythrocyte plasma membrane remained observable until the final stage of the breakdown process. Together with a peripheral layer of indigestible hemoglobin it might form a barrier for further penetration of lysosomal enzymes into the ingested erythrocyte. The hemoglobin of the erythrocyte is suggested to diffuse through the erythrocyte plasma membrane into the interspace between this membrane and the lysosomal membrane. Subsequently, the hemoglobin is digested in the interspace or in fragments pinched off from erythrocyte-containing lysosomes (=erythrolysosomes). The fragmentation of erythrolysosomes is considered to be the most efficient mechanism for the breakdown of red blood cells in the trophoblastic epithelium of the sheep placenta. The method of entry of hydrolytic enzymes into erythrocyte-containing phagosomes is discussed.     

Large Differences in Erythrocyte Stability Between Species Reflect Different Antioxidative Defense Mechanisms

We have developed a screening assay for erythrocyte stability, which is rapid, easy, inexpensive, robust, and suitable for handling a large number of samples in parallel. Erythrocytes are incubated overnight in 96-well microtiter plates in absence or presence of various oxidants, intact cells are pelleted by centrifugation, and lysis is determined by release of intracellular constituents into the supernatant as either activity of lactate dehydrogenase (LDH) or absorbance of hemoglobin at 406 nm. There is good correlation between the methods. A number of advantages by the present method are that only small amounts of blood is needed, washing is optional, erythrocytes may be stored for at least one day before assay, and large numbers of samples can be handled in parallel. Using this set-up, we have compared erythrocyte stability from several different animal species. We find that erythrocyte susceptibility towards lysis induced by H₂O₂ and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) is highly species dependent. The different susceptibility between species is due to cellular components, since swapping of plasma between species has little or no effect. As a novel observation, we find that erythrocytes from chicken are the most sensitive of the species tested towards lysis by H₂O₂ and are almost four orders of magnitude more sensitive than erythrocytes from man. This is due to a much lower content of catalase in erythrocytes from chicken. A more narrow range is observed for susceptibility towards AAPH and the ranking between the species is different. Thus, chicken erythrocytes are more resistant towards AAPH than some mammals by up to two orders of magnitude. This differential stability towards different oxidative stressors is likely due to evolution/selection of different defense mechanisms.     

Uptake of Lucifer yellow CH in leaves ofCommelina communis is mediated by endocytosis

Summary Lucifer yellow CH (LY) uptake into intact leaves ofCommelina communis has been studied with conventional fluorescence microscopy as well as confocal laser scanning microscopy. LY, a highly fluorescent tracer for apoplastic transport in plants and fluid phase endocytosis in animal cells, accumulates in the vacuole of leaf cells. However, considerable differences in the ability to take up LY were observed among the various cell types. Mesophyll cells take up large amounts of the dye whereas epidermal cells, including guard and subsidiary cells, showed no fluorescence in their vacuoles. An exception to this are trichome cells which show considerable accumulation of LY. When introduced into the cytoplasm of mesophyll protoplasts ofC. communis by means of a patch-clamp pipette, LY does not enter the vacuole. This supports the contention that exogenous LY can only gain access to the vacuole via endocytosis. Differences in the capacity for LY uptake may therefore reflect differences in endocytotic activity.Abbreviations CLSM Confocal laser scanning microscopy - DIC differential interference contrast - LY Lucifer yellow CH - PM plasma membrane     

Osmotic hemolysis and fragility A new model based on membrane disruption,and a potential clinical test

Red cell osmotic hemolysis has traditionally been defined by the loss of hemoglobin, in response to reduced osmotic pressure, as measured spectroscopically. Previous work from this laboratory using resistive pulse spectroscopy (RPS) has shown that in a mixed population of hemolyzing cell, ghosts can be detected as being more deformable, and hence appearing distinctly smaller, than the remaining intact cells. Other researchers using similar methods have reported detection of ghosts as apparently smaller objects, resulting from their greater sensitivity to dielectric breakdown. We now confirm both of these results, and demonstrate by kinetic studies that changes which occur in the rheological and electrical properties of ghosts are independent phenomena. We include in our analysis the explicit calculation of ghost and intact spherocyte resistivity after dielectric breakdown. The two different characterizations for ghosts are integrated into a proposed model of osmotic hemolysis based on known red blood cell membrane and cytoplasmic properties. This work provides both a theoretical and a practical foundation for RPS-based measures of osmotic fragility, including a potential new clinical test, measures which provide very early detection of the ultimate fate of osmotically stressed red cells.     

Observations on the ultrastructure of nucleated erythrocytes and thrombocytes,with particular reference to the structural basis of their discoidal shape

Summary The marginal band of nucleated erythrocytes in the toadfish is found, in electron micrographs, to be composed of about twenty-five microtubules approximately 200 Å in diameter. These form a bundle that encircles the erythrocyte just beneath the plasma membrane. These observations support the interpretation of Meves 1904, that this relatively stiff equatorial band may contribute to the maintenance of the discoid shape of nucleated erythrocytes in fish, amphibians, reptiles and birds.Similar microtubules form an annular bundle encircling the nucleus in fish thrombocytes. The number of tubular elements involved here is in excess of one hundred and they are located deep to the ectoplasmic layer instead of immediately beneath the plasmalemma. The term endoplasmic ring is therefore proposed for this structure.Comparative observations on nucleated erythrocytes of various species are presented showing that the density and fine structure of the material occupying the interchromosomal areas of the nucleus, always matches the cytoplasm and is related to the hemoglobin concentration of the species. These ultrastructural observations are consistent with the optical absorption and biochemical findings of other investigators indicating the presence of intranuclear hemoglobin in nucleated erythrocytes. Crystalline order is occasionally found in electron micrographs of the hemoglobin rich areas of the nucleus in toadfish erythrocytes but is not found in the cytoplasm.This research was supported by grant G-12916 of the National Science Foundation.     

Perspectives on hydrogen peroxide and drug-induced hemolytic anemia in glucose-6-phosphate dehydrogenase deficiency

G-6-PD-deficiency is a genetic disorder of erythrocytes in which the inability of affected cells to maintain NAD(P)H levels sufficient for the reduction of oxidized glutathione results in inadequate detoxification of hydrogen peroxide through glutathione peroxidase. Although a variety of free-radical species may be produced during the interaction of xenobiotic agents with erythrocytes and hemoglobin, the inability to destroy peroxides seems to be the hallmark of the disease. Colloid osmotic hemolysis is seldom observed in this disorder and it is possible that hydroxyl radicals derived from peroxide damage both lipid and protein constituents of the plasma membrane so that its intrinsic mechanical properties are altered. Erythrocytes with damaged membranes become less deformable and may be subjected to mechanical entrapment in the microcirculation. Ultimate recognition of damaged cell and sequestration by phagocytes leads to anemia.     

Augmented water binding and low cellular water content in erythrocytes of camel and camelids.

We investigated a link between hemoglobin primary structure, hemoglobin hydrophobicity-hydrophilicity, and erythrocyte water content in various mammalian species. Some hemoglobin molecules, particularly those of the camel and camelids, contain more charged amino acid residues and are more hydrophilic than the hemoglobins of human and a number of other mammalian species. To test the in vivo significance of these alterations of hemoglobin primary structure, we determined the osmotically unresponsive erythrocyte water fractions in mannit solutions of various osmolarities at 4 degreesC. Among the species investigated, the size of the osmotically unresponsive erythrocyte water fraction relates in a positive linear way to hemoglobin hydrophilicity. The extreme low total erythrocyte water content of camel erythrocytes (1.1-1.3 g water/g dry mass) may be explained by a comparatively high osmotically unresponsive erythrocyte water fraction. It is proposed that alterations of hemoglobin sequences of camel and camelids may be the part of a natural selection process aimed at protecting these animals against osmotic dehydration in arid environments.     

Fine structure of developing hagfish erythrocytes with particular reference to the cytoplasmic organelles

Cytological changes accompanying the maturation of erythrocytes in the “Pacific hagfish” (Eptatretus stoutii) were studied. Great numbers of immature and mitotically dividing red blood cells in the peripheral circulation of the hagfish appear to indicate that extensive differentiation and proliferation occurs in the blood stream of this animal. The immature erythrocytes contained mitochondria, Golgi membranes, centrioles, microtubules and a high density of ribosomes in the cytoplasm. Intermediate stages revealed lysosomes in the cytoplasm. With progressive differentiation the hagfish erythrocytes accumulate hemoglobin and lose most of their cytoplasmic organelles. The various cytoplasmic organelles are apparently lost through a degradation process brought about by lysosomal autolysis. The undigested products of degradation such as mitochondrial and other intercellular membranes are apparently extruded by way of the plasma membrane. The plasma membrane of young as well as mature erythrocytes display evidence of intense pinocytotic activity. The nucleolus undergoes a reduction in size with progressive maturation. The cytoplasm of mature erythrocytes consists predominantly of hemoglobin. An equatorial microtubular marginal band is identifiable in differentiating erythrocytes.     

Electrical hemolysis of human and bovine red blood cells.

The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50 per cent hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50 per cent hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be approximately 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.     

Electrical hemolysis of human and bovine red blood cells

Summary The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50% hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50% hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.     

Superoxide anion and drug-induced hemolysis.

Superoxide anion, either generated during the autooxidation of dihydroxyfumaria acid or by the interaction of 1,4-naphthoquinone-2-sulfonate and intracellular hemoglobin in red cells pretreated with the intracellular superoxide dismutase inhibitor, diethyldithiocarbamate, produces structural changes in red cells hemoglobin and hypotonic lysis. No evidence for lipid peroxidation was found in red cells exposed to either 1,4 naphthoquinone-2-sulfonate in the presence of diethyldithiocarbamate or to dihydroxyfumaric acid, although the membranes of these cells exposed to either 1,4 naphthoquinone-2-sulfonate in the presence of diethyldithiocarbamate or to dihydroxyfumaric acid, although the membranes of these cells retained a green pigment. These results suggest that superoxide anion reacts with cellular hemoglobin to form hemoglobin breakdown products which bind to the red cell membrane and thereby increase the osmotic fragility of the cell.     

Mechanical degradation of polyacrylamide solutions as a model for flow induced blood damage in artificial organs

Human or animal blood is normally used as a test fluid for the in vitro evaluation of hemolysis by artificial organs. However, blood has some disadvantages (large biological variability and problems with cleaning the devices). For that reason, we searched for a reproducible technical fluid with blood-like flow characteristics that exhibits similar shear depending destruction. In this study, a direct comparison between erythrocyte damage of bovine blood and shear-induced degradation of polyacrylamide solution is given. A uniform shear field was applied to the fluids using a shear device with a plate-plate geometry. It was shown that similarities exist between erythrocytes disaggregation and breakdown of super molecular structures in polymer solutions, caused by mechanical stress. In both cases steady low shear viscositity was diminished and the elastic component of complex viscosity of blood and polymer solutions has been reduced. There is a correlation between shear-induced hemolysis of bovine blood and mechanical polymer-degradation, which depends on the applied shear stresses.     

The fatty acid composition of phospholipids and absorption spectra of lipid extracts from lamprey,frog, and rat erythrocytes

The work studies the content and fatty acid composition of phospholipids as well as the absorption spectra of lipid extracts from red blood cells of poikilothermal and homoiothermal animals at different evolutionary levels. The objects of study include two poikilothermal species, the river lamprey (Lampetra fluviatilis) that uses oxygen dissolved in water, and the common frog (Rana temporaria) that consumes oxygen both from water and from air. A homoithermal animal is the white rat (Rattus rattus) that inhabits the terrestrial-aerial environment. The animals are studied in winter and spring. The phospholipid content in lamprey blood plasma is found to be twice higher than that in its erythrocytes. In the frog and the rat, the ratio is reverse. Determination of the fatty acid lipid composition of red blood cell phospholipids suggests that membranes in the lamprey are denser than in the frog. As for the fatty acids in the erythrocyte fraction of rat blood, they appear to be less diverse, with a double prevalence of saturated acids over unsaturated ones and devoid of long chain (C22) ω3 fatty acids. All of this results in a lower degree of unsaturation and a denser packing of fatty acids in the membrane structures of rat erythrocytes. The mechanism of reversible binding of O₂ molecules to hemoglobin in erythrocytes is discussed. Presumably, the mechanism of interaction between molecules of O₂ and molecules of water prevents the exchange interaction of electrons of the hemoglobin iron atoms with an oxygen molecule. This is confirmed by our obtained absorption spectra, which show that in the lipid extract almost totally devoid of water the heme isolated from erythrocytes is converted to hemin.     

Hemagglutination and hemolysis by : lichen extracts

Twenty-two species of lichens from 10 different genera possessed a hemagglutinin for one or more of human, sheep, horse, cow, rabbit, guinea pig, and chicken erythrocytes. Hemolysins were also detected occasionally, but these were only active at low dilutions. In those species tested, the hemolytic principle was dialyzable; the hemagglutinating agent was not. Preliminary studies have indicated that the lichen hemagglutinins are nonspecific.     

Thermohemolysis of erythrocytes in the temperature range including physiologic (autohemolysis)]

The activation energy of thermohemolysis of erythrocytes changes from 36 +/- 5 kcal/mol (35-45 degrees C) to 97 +/- 5 kcal/mol (45-55 degrees C) at the temperature about 45 degrees C in isotonic buffer. The break on Arhenius' plot is preserved also when erythrocytes are placed into plasma. The character of Arhenius' plot is the same when erythrocyte hemoglobin is totally oxidated into methemoglobin by chemical way, though thermal stability of such erythrocytes is decreased. The scheme is presented in which thermohemolysis of erythrocytes occurs by two independent ways: thermodenaturation of hemoglobin (limiting stage of the process when t greater than 45 degrees C) and modification of membrane proteins by hemin, the last being a product of hemoglobin oxidation (limiting stage of the process when t less than 45 degrees C).     

Measurement and analysis of the shape recovery process of each erythrocyte for estimation of its deformability using the microchannel technique: the influence of the softness of the cell membrane and viscosity of the hemoglobin solution inside the cell

This study tried to evaluate the deformability of each erythrocyte by measuring the time constant of shape recovery just after the erythrocytes left the microchannels. We fabricated a microchannel array with a 5μm-square, 100μm-long cross-section on a PDMS sheet. Three different kinds of blood samples were prepared—healthy erythrocytes as a control, artificially membrane-hardened erythrocytes and artificial hemoglobin solution-diluted erythrocytes—to investigate the influence of erythrocyte's mechanical property changes on the time constant of shape recovery. These shape recovery processes were modeled and analyzed by a standard liner solid model. As a result, the temporal variation of the compressive strain of all erythrocytes showed exponential decay with time elapsed like a first order lag system, so the time constant of shape recovery could be calculated from the semi-logarithmic relaxation curve. The stiffer the cell membrane was using glutaraldehyde, the shorter the time constant for relaxation became compared to healthy erythrocytes. The diluted hemoglobin erythrocytes snapped back quicker than healthy ones. In addition, the time constant of healthy blood drawn from females was clearly shorter than that collected from males. However, the time constant of fully hemoglobin substituted erythrocytes was not affected by gender difference. These results indicate that there is not a significant difference in the stiffness of healthy cell membranes regardless of individual and gender differences. On the other hand, the viscosity of the hemoglobin solution inside the cell is one of the significant factors affecting the time constant. Therefore, these results suggest that the deformability of individual erythrocytes can be quantitated by the time constant for relaxation measured by microchannel techniques.     

Is flight architecture determined by physical constraints or by natural selection?: the case of the midge Chironomus plumosus

Males of the midge Chironomus plumosus fly solely to mate. They maintain station for long periods in moving air. Females patrol in search of males and, after receiving a spermatophore, fly to oviposition sites. The requirements of flight in males and females are therefore fundamentally different. Females are larger than males, on average, so these differences could stem from scaling rules governing the geometry of space. The same explanation might apply to flight differences within the sexes and even to peculiarities of flight architecture in C. plumosus compared to other flying animals. In other words, might flight design be accounted for entirely by mechanical constraints without recourse to natural selection?
To test this hypothesis, the power output of C. plumosu was measured as size-specific muscle mass. Contrary to expectation, little evidence was found of scaling effects in this measure of power. Despite its being among the smallest of animals to fly, C. plumosus turns out to have the largest mass of flight muscle, relative to body size, yet found among animals. Differences both between C. plumosus and other species and within C. plumosus are, in general, more readily accounted for by the requirements of the mating system within the viscous universe encountered by small flying animals. We conclude that it is natural selection rather than mechanical constraint that is the primary influence determining the architecture of flight in this small animal.