Osmotic and diffusive properties of intracellular water in camel erythrocytes: effect of hemoglobin crowdedness

Camel erythrocytes have exceptional osmotic resistance and is believed to be due to augmented water-binding associated with the high hydrophilicity of camel hemoglobin. In practical terms this means that the proportion of osmotically non-removable water in camel erythrocytes is nearly 3-fold greater than that in human erythrocytes (approximately 65 vs approximately 20%). The relationship between water diffusion and the osmotic characteristics of intracellular water is the subject of this report. The amount of osmotically inactive water is 2-fold greater in camel hemoglobin solution in vitro compared to that of human, but water diffusion does not differ in camel and human hemoglobin solutions. However, the evaluation of water diffusion by magnetic resonance measurements in camel erythrocytes revealed approximately 15% lower apparent diffusion coefficient (ADC) compared with human erythrocytes. When human erythrocytes were dehydrated to the level of camel erythrocytes, their osmotic and water diffusion properties were similar. These results show that a lower ADC is associated with a more pronounced increase in osmotically inactive water fraction. It is proposed that increased hemoglobin hydrophilicity allows not only augmented water-binding, but also a closer hemoglobin packaging in vivo, which in turn is associated with slower ADC and increased osmotic resistance.     

On the osmotically unresponsive water compartment in cells

Differences in colligative properties (freezing point, boiling point, vapor pressure and osmotic behavior) between water in living cells and pure bulk water were investigated by re-evaluating reports of the osmotic behavior of mammalian cells. In five different animal cells, osmotically unresponsive water (OUW) values ranged from 1.1 to 2.2 g per g dry mass. Detailed analysis of human red blood cell (RBC) data indicates a major role for hemoglobin OUW-values, aggregation and packing in cell volume regulation that can be explained for the first time in relevant molecular terms.     

Steady-state volumes and metabolism-independent osmotic adaptation in mammalian erythrocytes

Erythrocytes of various mammalian species -- including human -- maintain osmotic balance with the blood plasma (osmotic activity 270-310 mosmol). However, their intracellular levels of osmotically active ions (potassium, sodium, chloride, and hydrogencarbonate), water content and osmotic resistance deviate significantly. In the present report we study the relationship among intracellular water, potassium and sodium levels of the erythrocytes of various mammalian species and in the developing calf. In addition, the osmotic resistance, K(+) (Rb(+)) uptake and the DPH fluorescence anisotropy of various erythrocytes and erythrocyte ghost membranes were correlated. The results show no statistically significant relationship between erythrocyte water content and [K(+)+Na(+)] levels or K(+)/Na(+) ratios. The reversal of erythrocyte K(+)/Na(+) ratios coincides with the decrease of steady-state ATP levels in the developing calf. The mobility of lipids within the hydrophobic inner layer of the plasma membrane relates closely to passive K(+) (Rb(+)) uptake, and plays a significant role in regulatory volume changes.     

Osmotically unresponsive water fraction on proteins: non-ideal osmotic pressure of bovine serum albumin as a function of pH and salt concentration

How much does protein-associated water differ in colligative properties (freezing point, boiling point, vapor pressure and osmotic behavior) from pure bulk water? This question was approached by studying the globular protein bovine serum albumin (BSA), using changes in pH and salt concentration to alter its native structural conformation and state of aggregation. BSA osmotic pressure was investigated experimentally and analyzed using the molecular model of Fullerton et al. [Biochem Cell Biol 1992;70(12):1325]. Analysis yielded both the extent of osmotically unresponsive water (OUW) and the effective molecular weight values of the membrane-impermeable BSA solute. Manipulation of BSA conformation and aggregation by membrane-penetrating cosolutes show that alterations in pH and salt concentration change the amount of bulk water that escapes into BSA from a minimum of 1.4 to a maximum of 11.7 g water per g dry mass BSA.     

Effects of dehydration on water relations and survival of lumbricid earthworm egg capsules

Earthworm egg capsules of five species were compared with regard to survival and water relations upon exposure to controlled dehydration at 20°C. Cocoons of the investigated species all contained about 3.5 g water·g^-1 dry weight when fully hydrated. Approximately 18% of this does not readily freeze upon cooling to -40°C and is referred to as osmotically inactive water. Cocoons exposed to desiccation lose a large proportion of the osmotically active water over 1–4 days until water in the cocoon fluids has equilibrated with surrounding water vapour. The amount of osmotically inactive water, on the other hand, is only reduced by 10–20%. Dendrobaena octaedra was the species most tolerant to drought, its tolerance limit coinciding with loss of practically all osmotically active water. For the five species investigated, there seemed not to be any clear correlation between drought tolerance and microhabitat. Previous investigations have suggested a very close relation between tolerance to dehydration and to subzero temperatures in overwintering earthworm cocoons. Survival at a given level of dehydration at room temperature is less than at temperatures below 0°C, and the tolerance of room temperature dehydration is not closely correlated with cold hardiness across the range of the species studied.Abbreviations dw dry weight - DSC differential scanning calorimetry - fw _pd fresh weight of partially dehydrated cocoons - OAW osmotically active water - OIW osmotically inactive water - Osm osmolality - water potential - R universal gas constant - T absolute temperature - V specific volume of water     

A model to explain the osmotic pressure behavior of hemoglobin and serum albumin

Previously published osmotic pressure data on hemoglobin and bovine serum albumin were used to determine the osmotically unresponsive solvent volume per unit dry mass of protein. A model is presented that accounts for the osmotic pressure of globular proteins based on a surface-associated osmotically unresponsive solvent volume. The model also accounts for changes in the osmotically unresponsive solvent volume owing to changes in pH, cosolute salt concentration, protein conformation, and protein aggregation.     

Generation of peptides by human erythrocytes: facts and artifacts

Previously reported data on peptide composition of human erythrocyte lysate were obtained under conditions that did not exclude proteolytic degradation of hemoglobin in the process of peptide isolation. Comparative chromatographic analysis of the diluted erythrocyte lysate incubated in acidic conditions with or without proteolytic enzyme inhibitors showed that several peptides earlier identified as intraerythrocyte ones in fact result from hemoglobin degradation by erythrocyte acidic protease(s) during incubation of the lysate. A rational scheme excluding postlysis proteolysis was developed for isolation of peptide fraction. Further analysis resulted in determination of structure and content of about 50 endogenous intraerythrocyte hemoglobin fragments. A primary endopeptidase splitting of alpha- and beta-globin chains followed by consecutive exopeptidase trimming of primary fragments is suggested as a degradation mechanism. The intraerythrocyte peptides were shown to differ from peptides excreted by the erythrocytes to the extracellular medium in the primary culture. It was also found that intraerythrocyte peptides cannot play the role of precursors of hemoglobin fragments present in tissue extracts.     

Unique properties of the camel erythrocyte membrane: II. Organization of membrane proteins

Camel erythrocyte membranes are distinguished by some unique properties of stability and composition. Notable is their abundance in proteins (protein: lipid ratio of 3 : 1). Membrane proteins of camel erythrocytes were compared with those of human erythrocytes, which have been intensively investigated. Proteins were extracted with various aqueous media (EDTA, alkaline or high ionic strength) and with ionic and non-ionic detergents and were analyzed by gel electrophoresis. In membranes of camel erythrocytes, the peripheral proteins constitute, proportionally, a much smaller fraction of total proteins than in the human erythrocyte, while their distribution is identical per unit of surface area. The camel erythrocyte membrane is particularly rich in integral proteins and in intramembranous particles. The proteins in this membrane are more closely organized than in the human system, as revealed by crosslinking and freeze-etching studies. It is proposed that protein-protein interaction of integral proteins, presumably constituting an “integral skeleton”, is a dominant structural feature stabilizing the camel erythrocyte membrane.     

Oxygen transfer properties and dimensions of red blood cells in high-altitude camelids,dromedary camel and goat

Summary To estimate the advantage of the small red blood cells (RBC) of high-altitude camelids for O₂ transfer, the kinetics of O₂ uptake into and release from the RBC obtained from llama, vicuña and alpaca were investigated at 37°C with a stopped-flow technique. O₂ transfer conductance of RBC (G) was estimated from the rate of O₂ saturation change and the corresponding O₂ pressure difference between medium and hemoglobin. For comparison, O₂ kinetics for the RBC of a lowaltitude camelid (dromedary camel) and the pygmy goat were determined and previously measured values for human RBC were used. O₂ transfer of RBC was found to be strongly influenced by extracellular diffusion, except with O₂ release into dithionite solutions of sufficiently high concentration (>30 mM). TheG values measured in these standard conditions,G _st (in mmol · min^–1 · Torr^–1 · (ml RBC)^–1) were: high-altitude camelids, 0.58 (averaged for llama, alpaca and vicuña since there were no significant interspecific differences); camel 0.42; goat, 0.42; man, 0.39. The differences can in part be attributed to expected effects of the size and shape of the RBC (volume, surface area, mean thickness), as well as to the intracellular O₂ diffusivity which depends on the concentration of cellular hemoglobin. The highG _st of RBC of highaltitude camelids may be considered to enhance O₂ transfer in lungs and tissues. But the O₂ transfer conductance of blood, , equal toG _st multiplied by hematocrit (in mmol · min^–1 · Torr^–1 · (ml blood)^–1), was only slightly higher as compared to other species: 0.20 (llama, alpaca, vicuña), 0.14 (camel), 0.18 (goat), 0.17 (man).Abbreviations DPG 2,3-diphosphoglycerate - G conductance - Hb hemoglobin - RBC red blood cells - percent saturation of hemoglobin     

Mammalian beta-adrenergic receptors. Structural differences in beta 1 and beta 2 subtypes revealed by peptide maps

Photoaffinity labeling techniques using p-azido-m-[125I]iodobenzylcarazolol have recently demonstrated that both the beta 1- and beta 2-adrenergic receptor-binding subunits from mammalian tissues including heart, lung, and erythrocytes reside on peptides of Mr approximately equal to 62,000-64,000. In this study, a two-dimensional gel electrophoresis method for peptide mapping was used to investigate and compare the structure of beta 1 - and beta 2-adrenergic receptor subtypes. When the photoaffinity labeled Mr approximately equal to 62,000 peptides from the beta 2-adrenergic receptors of rat lung and erythrocyte are subjected to simultaneous proteolysis using Staphylococcus aureus V8 proteinase or papain, exactly the same peptide fragments are generated from each subunit. In contrast, when the Mr approximately equal to 62,000 peptide containing the beta 1-adrenergic receptor-binding subunit derived from the rat heart is proteolyzed simultaneously with the Mr approximately equal to 62,000 peptide containing the beta 2-adrenergic receptors from either lung or erythrocyte, the peptide fragments generated are distinctly different. Peptide maps of beta 1-adrenergic receptors from the myocardial tissue of different species (pig versus rat) yield slightly different maps while the maps derived from the beta 2-adrenergic receptors of hamster lung and rat lung or erythrocytes reveal no interspecies differences. These data suggest: 1) alterations in the primary structure of the beta-adrenergic receptor may be responsible for the pharmacological specificities characteristic of beta 1- and beta 2-adrenergic receptor subtypes; and 2) alterations in the primary structure of similar beta-adrenergic receptor subtypes across different species may relate to the magnitude of their phylogenetic differences.     

Nuclear envelope fraction proteins: Isolation and comparison with the nuclear protein of the avian erythrocyte

A nuclear envelope fraction is obtained from circulating erythrocytes of mature chickens. This fraction was characterized by chemical analysis, ultramicroscopic examination and electrophoretic analysis of the polypeptides. It is free of plasma membrane polypeptides, of hemoglobin and of deoxyribonucleohistone. Comparisons of the sodium dodecyl sulfate-polyacrylamide-gel electropherograms of the nuclear envelope fraction polypeptides with either chromosomal fraction or the total nonhistone fraction polypeptides from Triton X-100-washed nuclei indicate that certain major polypeptides are common to all fractions. This indicates that many chicken erythrocyte nonhistone “chromosomal” polypeptides may be primarily associated with nuclear structural elements which are not, in fact, deoxyribonucleohistone. Although this finding cannot be extended per se to other cells, it does suggest that caution should be exercised in describing nuclear components as “chromosomal” when this designation is intended to imply primary association with the deoxyribonucleohistone. This analysis of the polypeptides in the nuclear envelope fraction of the erythrocyte indicates that further study of the envelope fraction will provide new insights into nuclear structure and function.     

Osmotic properties of ehrlich ascites tumor cells during the cell cycle

Ehrlich ascites tumor cells were grown and maintained in continuous spinner culture. The population of dividing cells was synchronized by a double thymidine block technique. Cell cycle phases were determined graphically by plotting mitotic index, cell number, and DNA synthesis against time. Changes in the osmotic properties of Ehrlich ascites tumor cells during the cell cycle are described. Permeability to water is highest at the initiation of S and progressively decreases to its lowest value just after mitosis. Heats of activation for water permeability vary during the cell cycle, ranging from 9–14 kcal/mole. Results may imply changes in the state of water in the membrane during the cycle. The volume of osmotically active cell water is highest during S and early G2 and decreases during the mitotic phase, as cells undergo division. Total water content remains stable at 82% (w/w) during the cycle. Total concentration of the three major ions (Na, K, Cl), expressed as mEq/liter total cell volume, does not change. The fraction of total cell water which is osmotically active (Ponder's R) decreased gradually from 0.75 at S to about 0.56 following mitosis. Findings suggest that a fraction of the total water within the cell exists in a “bound” form and is, therefore, incapable of being shifted under the driving force of osmotic pressure. This fraction of bound water increases during the cell cycle. Possible alterations in membrane fluidity and the state of water in the cell are discussed.     

The entrance of water into beef and dog red cells

The rate constants for diffusion of THO across the red cell membrane of beef and dog, and the rate of entrance of water into the erythrocytes of these species under an osmotic pressure gradient have been measured. For water entrance into the erythrocyte by diffusion the rate constants are 0.10 ± 0.02 msec.^-1 (beef) and 0.14 ± 0.03 msec.^-1 (dog); the permeability coefficients for water entrance under a pressure gradient of 1 osmol./cm³ are 0.28 See PDF for Equation These values permit the calculation of an equivalent pore radius for the erythrocyte membrane of 4.1 A for beef and 7.4 A for dog. In the beef red cell the change in THO diffusion due to osmotically produced cell volume shifts has been studied. The resistance to THO diffusion increases as the cell volume increases. At the maximum volume, (1.06 times normal), THO diffusion is decreased to 0.84 times the normal rate. This change in diffusion is attributed to swelling of the cellular membrane.     

Studies on camel hemoglobin. 1. Physico-chemical properties and some structural aspects of camel hemoglobin (Camelus dromedarius).

Hemoglobin from an adult camel (Camelus dromedarius) was prepared from the red cell lysate by CM- and DEAE-cellulose chromatography. The purified hemoglobin showed a lesser mobility on starch gel electrophoresis at pH 8.5 than that of human hemoglobin C. Native camel hemoglobin contains 95-99% alkali-resistant hemoglobin and in soluble in 2.94 M K2HPO4/KH2PO4 buffer. Different forms of camel hemoglobin show similar ammonium sulfate precipitation curves. Indirect evidence for the stability of camel hemoglobin solutions was obtained from several sources. Spontaneous met-hemoglobin formation is extremely slow and minimal quantities of degradation products appear on starch gel electrophoresis and on chromatographic separation. The alpha and beta chains of camel hemoglobin A were separated on a CM-23 column by the use of a pyridine formate gradient. Large peptide fragments were obtained by tryptic digestion of maleylated alpha and beta chains. The N-terminal structure of the alpha and beta chains and of tryptic maleylated peptides derived from alpha and beta chains are presented. Between adult camel hemoglobin and adult human hemoglobin six amino acid differences in the N-terminal 20 amino acid residues of the alpha chain, at residues: 4, 5, 12, 14, 17, and 19; eight amino acid substitutions were found in the beta chain at positions: 4, 5, 6, 9, 12, 13, 16, and 19. Substitutions at alpha5 Ala leads to Lys, and beta19 Asn leads to Lys, increase the net positive charge of camel hemoglobin by two, while other substitutions result in no charge differences. The molecular basis of the stability of camel adult hemoglobin is discussed.     

Effects of drought on water relations and nonstructural carbohydrates in cladodes of Opuntia ficus-indica

Nocturnal acid accumulation, water content, osmotic pressure (π), and nonstructural carbohydrates were determined in the chlorenchyma and the water-storage parenchyma of Opuntia ficus-indica (L.) Miller for well-watered plants and those subjected to drought for 15 weeks. During the 15-week drought, total cladode water content decreased by 57%, the water-storage parenchyma losing a greater fraction of water than the chlorenchyma, which most likely helped maintain nocturnal acid accumulation in the latter tissue. Despite the preferential water loss from the water-storage parenchyma, it had a lower π than the chlorenchyma over the 15 weeks of drought, suggesting a substantial decrease in osmotically active solutes in the water-storage parenchyma. Also, the measured π increases of both tissues were much less than those predicted based on the loss of water during drought and the initial content of osmotically active solutes under well-watered conditions. A decrease in the amount of soluble sugars (glucose. fructose and sucrose) occurred in plants subjected to drought. accounting for 46% and 81% of the difference between the measured and the predicted increases in π of the chlorenchyma and the water-storage parenchyma. respectively. The decrease in soluble sugars was associated with an equivalenl increase in polysaccharides, presumably starch, in the water-storage parenchyma. but not in the chlorenchyma.     

Fine structure of camel erythrocytes in relation to its functions

The fine structure of the red cells in Arabian camels was investigated and certain characteristic features were noted. The plasmalemma of camel erythrocytes are tri-laminar, the inner and outer membranes are of high electron density between which is a zone of lesser electron density. No intracellular organelles were observed with the occasional exception of a small number of mitochondria. In the camel erythrocyte, a marginal band consisting of 30-45 microtubules was observed in many cells. Some of the possible functions of the marginal band in camel erythrocytes are discussed.     

Wood day capacitance is related to water content,wood density,and anatomy across 30 temperate tree species

Water released from wood during transpiration (capacitance) can meaningfully affect daily water use and drought response. To provide context for better understanding of capacitance mechanisms, we investigated links between capacitance and wood anatomy. On twigs of 30 temperate angiosperm tree species, we measured day capacitance (between predawn and midday), water content, wood density, and anatomical traits, that is, vessel dimensions, tissue fractions, and vessel–tissue contact fractions (fraction of vessel circumference in contact with other tissues). Across all species, wood density (WD) and predawn lumen volumetric water content (VWC_L-pd) together were the strongest predictors of day capacitance (r²_adj = .44). Vessel–tissue contact fractions explained an additional ~10% of the variation in day capacitance. Regression models were not improved by including tissue lumen fractions. Among diffuse-porous species, VWC_L-pd and vessel–ray contact fraction together were the best predictors of day capacitance, whereas among semi/ring-porous species, VWC_L-pd, WD and vessel–fibre contact fraction were the best predictors. At predawn, wood was less than fully saturated for all species (lumen relative water content = 0.52 ± 0.17). Our findings imply that day capacitance depends on the amount of stored water, tissue connectivity and the bulk wood properties arising from WD (e.g., elasticity), rather than the fraction of any particular tissue.     

Shape changes in human erythrocytes induced by replacement of the native phosphatidylcholine with species containing various fatty acids

Phosphatidylcholine-specific transfer protein from beef liver has been used to replace native phosphatidylcholine (PC) molecules from intact human erythrocytes by a variety of PC species differing in fatty acid composition. These replacements changed neither the total phospholipid content of the membrane, nor the composition of this fraction in terms of the various phospholipid classes. The morphology of the erythrocyte was not modified when native PC was replaced by 1-palmitoyl,2-oleoyl PC, 1-palmitoyl,2-linoleoyl PC, egg PC, or PC isolated from rat liver microsomes. Replacement with the disaturated species 1,2-dimyristoyl PC, 1,2-dipalmitoyl PC, and 1,2-distearoyl PC resulted in the formation of echinocytes and, at higher levels of replacement, in spheroechinocytes. Echinocyte-like erythrocytes were also observed after replacement with 1-palmitoyl,2-arachidonoyl PC, whereas stomatocytes were formed upon replacement with PC species containing two unsaturated fatty acids, e.g., 1,2-dioleoyl PC and 1,2-dilinoleoyl PC. The observations show that the erythrocyte membrane structure and the overall discoid cell shape of the human erythrocyte are optimally stabilized by PC species that contain one saturated and one mono- or diunsaturated fatty acid, and that the cell tolerates only limited variations in the species composition of its PC.     

Soft X-ray microscopy analysis of cell volume and hemoglobin content in erythrocytes infected with asexual and sexual stages of Plasmodium falciparum

Plasmodium falciparum, the most virulent agent of human malaria, undergoes both asexual cycling and sexual differentiation inside erythrocytes. As the intraerythrocytic parasite develops it increases in size and alters the permeability of the host cell plasma membrane. An intriguing question is: how is the integrity of the host erythrocyte maintained during the intraerythrocytic cycle? We have used water window cryo X-ray tomography to determine cell morphology and hemoglobin content at different stages of asexual and sexual differentiation. The cryo stabilization preserves native structure permitting accurate analyses of parasite and host cell volumes. Absorption of soft X-rays by protein adheres to Beer–Lambert’s law permitting quantitation of the concentration of hemoglobin in the host cell compartment. During asexual development the volume of the parasite reaches about 50% of the uninfected erythrocyte volume but the infected erythrocyte volume remains relatively constant. The total hemoglobin content gradually decreases during the 48 h cycle but its concentration remains constant until early trophozoite stage, decreases by 25%, then remains constant again until just prior to rupture. During early sexual development the gametocyte has a similar morphology to a trophozoite but then undergoes a dramatic shape change. Our cryo X-ray tomography analysis reveals that about 70% of the host cell hemoglobin is taken up and digested during gametocyte development and the parasite eventually occupies about 50% of the uninfected erythrocyte volume. The total volume of the infected erythrocyte remains constant, apart from some reversible shrinkage at stage IV, while the concentration of hemoglobin decreases to about 70% of that in an uninfected erythrocyte.     

Molecular characterization of Streptococcus equi subspecies equi isolated from an Ethiopian camel by ribotyping and PCR-ribotyping.

The aim of this study was to characterize a S. equi subspecies equi strain isolated from an Ethiopian camel by different molecular techniques (Ribotyping and PCR-Ribotyping). We compared the results obtained with those generated from two strains of the Pasteur Collection. The ribotyping showed the highest power of differentiation, distinguishing between the strains analyzed, whereas PCR-Ribotyping was able only to differentiate the camel isolate but not the strains from the Pasteur Collection. The application of this technique will be very useful to establish a clonal relationship among equine and camelids strains and help the prevention and cure of the equine and camel pathology.