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 prevention of erythrocyte swelling upon dilution after freezing and thawing

Cellular swelling of erythrocytes exposed to Me2SO during freezing and thawing may lead to hemolysis upon dilution of the cryoprotectant with pure electrolyte buffer. Excessive cell swelling is effectively avoided by exposing the RBC to the nonpenetrating sorbitol after thawing and before dilution. Due to the initial reduction in volume by sorbitol, cell swelling upon dilution may not cause hemolysis particularly with concentrations of 0.05 to 0.15 M of sorbitol in the diluting electrolyte buffer. Membrane damage incurred during freezing and thawing is particularly pronounced with the older red cell population, while the younger population membrane integrity can be preserved to an optimal degree.     

Osmotic loading of in situ chondrocytes in their native environment

Changes in the osmotic environment cause changes in volume of isolated cells and cells in tissue explants, and the osmotic environment becomes hypotonic in cartilage diseases such as osteoarthritis (OA). However, it is not known how cells respond to a hypotonic osmotic challenge when situated in the fully intact articular cartilage. A confocal laser scanning microscope was used to image chondrocytes of intact rabbit patellae in an isotonic (300 mOsm) and hypotonic (172 mOsm) immersion medium. Cell volumes were calculated before and 5, 15, 60, 120 and 240 minutes after the change in saline concentration. Local tissue strains and swelling of the entire tissue were estimated from the relative movements of cells and displacements of single cells, respectively. Cell volumes increased rapidly (< or = 5 minutes, p<0.05) by approximately 22%, after which they remained constant for an hour (p>0.05). However, two and four hours post the hypotonic challenge, cell volumes were statistically greater (p<0.05) than those at all earlier time points, and swelling of the entire tissue continued throughout the four hour loading period. The results of our study suggest that osmotic loading induced volume changes of in situ chondrocytes in their native environment occur quickly and continue for hours. Understanding the behaviour of cells in their native environment provides novel insigth into the cell mechanics in ostearthritic joints and so may help understand the onset and progression of this disease.     

Alkaline hemolysis fragility is dependent on cell shape: results from a morphology tracker.

BACKGROUND: The morphometric analysis of red blood cells (RBCs) is an important area of study and has been performed previously for fixed samples. We present a novel method for the analysis of morphologic changes of live erythrocytes as a function of time. We use this method to extract information on alkaline hemolysis fragility. Many other toxins lyse cells by membrane poration, which has been studied by averaging over cell populations. However, no quantitative data are available for changes in the morphology of individual cells during membrane poration-driven hemolysis or for the relation between cell shape and fragility. METHODS: Hydroxide, a porating agent, was generated in a microfluidic enclosure containing RBCs in suspension. Automatic cell recognition, tracking, and morphometric measurements were done by using a custom image analysis program. Cell area and circular shape factor (CSF) were measured over time for individual cells. Implementations were developed in MATLAB and on Kestrel, a parallel computer that affords higher speed that approaches real-time processing. RESULTS: The average CSF went through a first period of fast increase, corresponding to the conversion of discocytes to spherocytes under internal osmotic pressure, followed by another period of slow increase until the fast lysis event. For individual cells, the initial CSF was shown to be inversely correlated to cell lifetime (linear regression factor R=0.44), with discocytes surviving longer than spherocytes. The inflated cell surface area to volume ratio was also inversely correlated to lifetime (R=0.43) but not correlated to the CSF. Lifetime correlated best to the ratio of cell inflation volume (Vfinal-Vinitial) to surface area (R=0.65). CONCLUSIONS: RBCs inflate at a rate proportional to their surface area, in agreement with a constant flux model, and lyse after attaining a spherical morphology. Spherical RBCs display increased alkaline hemolysis fragility (shorter lifetimes), providing an explanation for the increased osmotic fragility of RBCs from patients who have spherocytosis.     

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.     

Osmotic loading of articular cartilage modulates cell deformations along primary collagen fibril directions

Osmotic loading is known to modulate chondrocyte (cell) height, width and volume in articular cartilage. It is not known how cartilage architecture, especially the collagen fibril orientation, affects cell shape changes as a result of an osmotic challenge.Intact patellae of New Zealand white rabbits (n=6) were prepared for fluorescence imaging. Patellae were exposed to a hypotonic osmotic shock and cells were imaged before loading and 5–60 min after the osmotic challenge. Cell volumes and aspect ratios (height/width) were analyzed. A fibril-reinforced poroelastic swelling model with realistic primary collagen fibril orientations, i.e. horizontal, random and vertical orientation in the superficial, middle and deep zones, respectively and cells in different zones was used to estimate cell aspect ratios theoretically.As the medium osmolarity was reduced, cell aspect ratios decreased and volumes increased in the superficial zone of cartilage both experimentally (p<0.05) and theoretically. Theoretically determined aspect ratios of middle zone cells remained virtually constant, while they increased for deep zone cells as osmolarity was reduced.Findings of this study suggest that osmotic loading modulates chondrocyte shapes in accordance with the primary collagen fibril directions in articular cartilage.     

Delayed hemolysis of human erythrocytes in solutions of glucose

There has been described a type of hemolysis which occurs under certain defined conditions when erythrocytes are suspended in glucose solution. It consists of a prolytic phase lasting about an hour, followed by a hemolytic phase lasting about 2 hours. The physical factors controlling this delayed hemolysis have been investigated. The system is especially sensitive to changes of pH and of temperature. This type of hemolysis is inhibited by increased osmotic pressure and by phlorhizin, but not, as far as can be ascertained, by fluoride or iodoacetate. It is possible, but not yet proved, that delayed hemolysis in glucose solution is dependent on enzymic activity. Phosphorylation may be the limiting factor. During the prolytic phase the cells are easily permeable to potassium. It is concluded that the development of cation permeability is not a direct cause of hemolysis.     

SEASONAL CHANGES IN STRUCTURE OF A SUBMERGED BLUEGREEN ALGAL/BACTERIAL COMMUNITY FROM A GEOTHERMAL HOTSPRING1

The species (cytospecies) structure and seasonal dynamics of a thermophilic bluegreen algal/bacterial community from Mimbres, New Mexico are described. Using stereological techniques, several common quantitative ecological parameters (biomass, density, distribution measurements, Species Diversity Indices, importance values) were described for the community growing ca. 1 m beneath the water surface on the inner walls of a concrete cistern. Temperature and pH were relatively constant while water flow rates, duration and intensity of light showed seasonal variation. The total volume of the mat occupied by living organisms did not exceed 2% during the entire year. The remaining volume was occupied by gas and water bubble chambers, gelatinous matrix, empty tubes, mineral deposits, and water. The total biomass remained constant except during a period of severe, seasonal light reduction (November). Species Diversity Indices, density and importance values for the bluegreen algal and bacterial populations showed seasonal changes. The three dominant bluegreen algal cytospecies which represented the greatest amount of biomass alternated in their abundance on a seasonal basis while the three dominant bacterial cytospecies remained the same throughout the period of study. Although considerable season change occurred in the species structure of this community, the total living volume remained relatively constant with the major changes in the bluegreen algal populations. Many of the seasonal changes observed appear to correlate with seasonal changes in light intensity and duration.     

Morphological alterations and cytoskeletal reorganization in opossum kidney (OK) cells during osmotic swelling and volume regulation

Cells from a variety of tissues regulate their volume when exposed to anisotonic conditions. After exposure of cells to hypotonic conditions, the rapid phase of cell swelling is followed by a slower phase of cell shrinkage towards the initial volume. The present study investigates morphological alterations of adherent and fully spread cells after exposure to hypotonic conditions and the reorganization of cytoskeletal components such as F-actin, actin-binding proteins, microtubules and intermediate-sized filaments. We used cells of a continuous epithelial cell line from the opossum kidney (OK cells), which were exposed to hypotonic conditions for a period of 60 min at 25° C. The osmolarity was reduced by 40% from 320 mosmol/l (isotonic conditions) to 192 mosmol/l (hypotonic conditions). The initial swelling after exposure of OK cells to hypotonic conditions caused enhanced ruffling membrane activity, formation of lamellipodia and an extended space between adjacent cells which was caused by a more rounded cell shape. Moreover, the height of cells located in the centre of cell clusters increased by 32±8% (mean value±SEM) as checked by morphometric analysis of the vertical distance between the apical and basolateral F-actin domain. Although the fluorescence intensity and organization of F-actin in a horizontal direction remained unaltered during cell swelling, we observed a loss of periodicity and irregular distribution of myosin aggregates and a partial rear-rangement of vimentin filaments in the form of short fragments. In all experiments the organization of microtubules was observed to be unaltered. The alterations described above were reversible during cell shrinkage towards the initial volume, i.e. at 60 min after exposure to hypotonic conditions cell morphology and cytoskeletal organization no longer differed from the corresponding controls which were kept under isotonic conditions for the whole experimental period. The results demonstrate that only certain intracellular cytoskeletal components are actively involved in cell swelling and regulatory volume decrease.     

Time dependent modifications of Hep G2 cells during exposure to static magnetic fields

Morphological modifications, i.e., cell shape, cell surface sugar residues, cytoskeleton, and apoptosis of Hep G2 cells during 24 h exposure to 6 mT static magnetic field (static MF) were studied by means of light and electron microscopy and cytochemistry. Progressive modifications of cell shape and surface were observed during the entire period of exposure to static MF. Control cells were polyhedric with short microvilli covering the cell surface, while those exposed to static MF, were elongated with many irregular microvilli randomly distributed on the cell surface. At the end of the exposure period, the cells had a less flat shape due to partial detachment from the culture dishes. However, throughout the period of exposure under investigation, the morphology of the organelles remained unmodified and cell proliferation was only partially affected. In parallel with cell shape changes, the microfilaments and microtubules, as well as the quantity and distribution of surface ConA-FITC and Ricinus communnis-FITC labeling sites, were modified in a time dependent manner. Apoptosis, which was almost negligible at the beginning of experiment, increased to about 20% after 24 h of continuous exposure. The induction of apoptosis was likely due to the increment of [Ca2+]i during exposure. In conclusion, the data reported in the present work indicates that 6 mT static MF exposure exerts time dependent biological effects on Hep G2 cells.     

Content of Adenosine Phosphate Compounds in a Long-Day Duckweed, Lemna gibba G3, under Different Light and Nutritional Conditions

The levels of ATP ADP and AMP as well as the energy charge were examined in the long-day duckweed. Lemna gibba G3, under different light and nutritional conditions. ATP and ADP content, but not AMP, decreased slightly when the plant was cultured with a medium depleted of sucrose or was exposed to continuous darkness. The energy charge was not affected either by changes in the light conditions or by the depletion of sucrose from the medium, although increase of fresh weight changed drastically under these conditions. The levels of ADP and AMP and the value of the energy charge remained nearly constant throughout a 1-day period when the plant was exposed to short-day, continuous light and continuous dark. ATP content decreased gradually during the light period of the short day and thereafter remained constant during the dark period. When the plant was exposed to continuous light. ATP content decreased until the 8th hour after beginning of the continuous light, only to recover rapidly to its original level during the phase coinciding with the preceding dark period of the short day. Under continuous dark conditions the ATP content remained constant throughout the day. It was concluded that the diurnal rhythm of physiological activities previously reported cannot be related to the energy charge.     

Cell volume increase in Escherichia coli after shifts to richer media.

Synchronous cultures of Escherichia coli 15-THU and WP2s, which were selected by velocity sedimentation from exponential-phase cultures growing in an acetate-minimal salts medium, were shifted to richer media at various times during the cell cycle by the addition of glucose or nutrient broth. Cell numbers and mean cell volumes were measured electronically. The duration of the division cycle of the shifted generation was not altered significantly by the addition of either nutrient. Growth rates, measured as rates of cell volume increase, were constant throughout the cycle in unshifted acetate control cultures. When glucose was added, growth rates also remained unchanged during the remainder of the cell cycle and then increased abruptly at or after cell division. When nutrient broth was added, growth rates remained unchanged from periods of 0.2 to 0.4 generations and then increased abruptly to their final values. In all cases, the cell volume increase was linear both before and after the growth rate transition. The strongest support for a linear cell volume increase during the cell cycle of E. coli in slowly growing acetate cultures, however, was obtained in unshifted cultures, in complete agreement with earlier observations of cell volumes at much more rapid growth rates. Although cell growth and division are under the control of the synthesizing machinery in the cell responsible for RNA and protein synthesis, the results indicate that growth is also regulated by membrane-associated transport systems.     

Maturation and degeneration of the fat body in the Drosophila larva and pupa as revealed by morphometric analysis

Using morphometric and cytochemical techniques we have described changes taking place in the fat body cells during three different stages of development. The cell number remains constant at about 2200 cells during larval life and then decreases gradually and continuously throughout metamorphosis and the first 3 days of the adult stage until no more cells can be observed. Cell size increases rapidly during the larval period and decreases steadily during metamorphosis and adult stage. The size of the nuclei increases during the larval instars and decreases during the pupal interval. The change in nuclear size is correlated with the amount of DNA present throughout development implying the nuclear DNA is synthesized during the larval period and degraded gradually during metamorphosis. The cell size changes are due in large part to accumulation or loss of reserve substances: lipid droplets, glycogen deposits and protein granules. During metamorphosis the amount of lipid decreases slightly whereas glycogen experiences two loss cycles. The protein granules in the form of lysosomes continue to increase in amount during the first day of metamorphosis because of a short period of massive autophagy. Then the lysosomes decrease in amount throughout the remainder of metamorphosis. The lysosomes stain positively for lipofuscin.     

Seasonal variations in pituitary LH-gonadotropes of the hibernating bat Myotis lucifugus lucifugus: an immunohistochemical study

Pituitary gonadotropes were identified throughout the year in the seasonally breeding, hibernating bat Myotis lucifugus lucifugus by means of light microscopic immunohistochemistry. In both male and female bats, these cells were immunoreactive with an antiserum directed to the beta subunit of luteinizing hormone. Some gonadotropes were aggregated near a portion of the infundibular stalk which crosses the anterior lobe, while most were scattered singly in a uniform manner throughout the rest of the pars distalis. This cell population exhibited seasonal variations in both sexes. In males, the proportional volume of the pars distalis occupied by immunoreactive gonadotropes (volume fraction) was significantly reduced in late July, when plasma testosterone levels were approaching their seasonal peak. In females, the volume fraction declined in April, following ovulation, and remained low during pregnancy and lactation. The size and shape of gonadotropes appeared relatively constant throughout the annual reproductive cycle in male bats; the immunoreactive cells were irregular in shape, with cytoplasmic extensions insinuating between and often "cupping" other secretory cell types. In females, the gonadotropes resembled those of males throughout most of the year, except during pregnancy, when these cells became enlarged and ovoid. No evidence of involution was observed in these anterior pituitary cells in either males or females during hibernation.     

Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume,morphology, rheology and hemolysis

Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis.We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry.Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3–36-fold) and hemolysis (1–44-fold), partially compensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells. Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43–67%).The un-acylated toxin ProHlyA and the deletion analog HlyA∆914-936 were unable to induce ATP release or hemolysis.For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure.HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPe activation of purinergic P2X receptors reduced swelling by 60–80%. HlyA-RBCs showed an acute 1.3–2.2-fold increase of Ca²⁺i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media.Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity.     

Hypotonic hemolysis of human red blood cells: a two-phase process.

Previous use of hemolysis time measurement to determine permeability coefficients for the red blood cell membrane rested on the assumption that cells swelling in a hypotonic medium hemolyzed immediately on reaching critical volume. By preswelling red cells to various volumes prior to immersion in hemolytic solutions we extrapolate to the hemolysis time of red cells immersed at critical volume and thereby find a significant period of time during which the cells apparently remain in a spherical form prior to release of hemoglobin. Revised estimates of permeability coefficients follow from including this spherical (nonswelling) phase. In addition, the appreciation of a characteristic time period during which the membrane is under tension provides new opportunity to study physical and chemical properties of the membrane.     

Changes in mammary secretory epithelial reactions to oxytocin action under the influence of ouabain

Changes in shape and volume of mammary secretory cells and in their RNA content, under the action of oxytocin, are considered. Under ouabain suppression of Na,K-ATPase activity, no swelling or shape changes occur in the secretory cells, in addition to no increase in their RNA content, which is characteristic of the cell reaction during the initial period of oxytocin action.     

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.     

Transformation frequency increases with increase in agitation rate of chemostat-cultivated Escherichia coli K12, strain C-600

The transformation frequency of Escherichia coli C-600, continuously cultivated in a chemostat operated at constant dilution rate, increased with increase in agitation rate (impeller speed). Cell counts at each impeller speed remained approximately constant. The phenomenon correlated with changes in mean cell volume associated with the changes in agitation rate.     

Volume changes in hemolytic systems containing resorcinol,taurocholate, and saponin

Simultaneous measurement of hemolysis, the volume of the intact cells, and the K lost from the intact cells of systems containing resorcinol, sodium taurocholate, and saponin shows that the volume increases may be conspicuously small while the K losses are large, and that the volume increases are un-equal for equal K losses produced by different lysins. In higher concentrations of the same lysins, the critical volume for hemolysis is a function of the nature of the lysin and of its concentration. It is impossible to say whether these observations are compatible with the current "dual mechanism" and "colloid osmotic" hypotheses of hemolysis, in which the swelling of the cell is supposed to result from the lysin having made it cation-permeable. The difficulty to be overcome is that the theory cannot be developed to describe volume changes in finite time unless we know what assumptions to make about the mobilities of K and Na, the forces driving them into and out of the cell, etc. The experimental results do not suggest, however, that any simple set of assumptions would be satisfactory. The conditions which regulate the upper limit of the swelling, i.e. the point at which a swelling phenomenon becomes a hemolytic phenomenon, are functions of the nature of the lysin and sometimes of its concentration. They require to be specified by an independent statement, over and above any statement which may be made about the rate at which swelling occurs in the system. The simplest view of the situation is that the conditions which regulate the critical volume and those which regulate the rate of swelling are both functions, as yet undefined, of the reaction which takes place between the lysin and the structural components of the red cell.