Dog Erythrocytes Research Articles

Genetic control of glycolipid expression

A polymorphic variation of sialic acid species of sialosyllactosylceramide was found in dog erythrocytes. The analysis of the glycolipids in the erythrocytes of the individual dogs in a family of a Japanese breed of dog, Shiba-Inu, showed that the expression of sialosyllactosylceramide containing N-glycolylneuraminic acid was an autosomal dominant trait over the expression of that containing N-acetylneuraminic acid. Polymorphic variations of major liver gangliosides were also found in various strains of inbred mice. The strains were classified into three groups; the first group possessed only II 3 Neu-Gc-LacCer, the second group possessed II 3NeuGc-GgOse 3Cer in addition to II 3NeuGc-LacCer and the third group possessed II 3NeuGc-GgOse 4Cer and II 3NeuGc,IV 3NeuGc-GgOse 4Cer as well as the above two gangliosides. By subjecting mice of these three groups to genetic analysis, the strain of the first group (WHT/Ht mice) was demonstrated to be a recessive homozygote which had a single autosomal defective gene making it unable to express N-acetylgalactosaminyltransferase activity to produce II 3NeuGc-GgOse 3Cer. The strains of the second group (BALB/c and C57BL/10 mice) were also demonstrated to be recessive homozygotes which had a single autosomal defective gene making them unable to express high enough level of galactosyltransferase activity to produce II 3NeuGc-GgOse 4Cer. By the analysis of gangliosides and the enzyme activity of H-2 congenic mice and mice produced by a mating, this defective gene controlling the expression of II 3NeuGc-GgOse 4Cer through the regulation of the transferase activity was demonstrated to be linked to H-2 complex on chromosome 17.

Relationship between membrane atpase and shape changes in the dog erythrocyte

The effects of heating blood to 57° C on intraerythrocytic calcium, membrane ATPase activity and cell shape have been studied in canine blood. Intraerythrocytic calcium was determined by use of arsenazo III, membrane ATPase activity was determined by inorganic phosphorous formation and erythrocyte shape was determined by scanning electron microscopy. The results of this study showed that this degree of thermal trauma would cause a 27% increase in intraerythrocytic calcium and a 38% decrease in ATPase activity. During these changes in calcium and ATPase activity the erythrocyte changed form from biconcave to spherical. Addition of catalase (3,200 U/ml) to the blood prior to heating prevented the changes observed in intraerythrocytic calcium, membrane ATPase activity and shape. The addition of the free-radical generating combination of hypoxanthine-xanthine oxidase to blood produced a 20% decrease in membrane ATPase activity and a change in erythrocyte shape, but did not alter intraerythrocytic calcium. These results suggest that free-radicals are responsible for the changes in membrane ATPase activity. The observation that shape change occurs when ATPase activity has been decreased, but calcium has not been increased, suggests that membrane ATPase activity levels are more important in producing changes in erythrocyte shape than are intraerythrocytic calcium levels.

Pyruvate kinase deficiency in dog and human erythrocytes: effects of energy depletion on cation composition and cellular hydration.

Pyruvate kinase-(PK) deficient human reticulocytes incubated 4 hr under conditions where mitochondrial oxidative phosphorylation is inhibited become ATP-depleted. Concomitantly, these energy-depleted red blood cells (RBC) lose massive amounts of potassium and water. PK-deficient reticulocyte-rich RBC from a Basenji dog, incubated under the same conditions, also manifest ATP-depletion and K-loss. In contrast to the cation changes in human cells, however, K-loss in PK-deficient dog reticulocyte-rich RBC is balanced by an equivalent Na gain, and consequently these canine erythrocytes do not undergo any change in cellular hydration. Potassium depletion and dehydration, which may be related to membrane injury in human RBC, do not appear to be involved in the hemolysis of PK-deficient dog erythrocytes.

The temperature dependence of passive potassium permeability in mammalian erythrocytes

The effect of temperature on the “passive” permeability of mammalian plasma membranes to K +, measured as the residual flux in the presence of ouabain and bumetanide, was investigated in erythrocytes of several species. Without Ca 2+ in the medium, only human red cells demonstrated the “paradoxical” rise in passive flux at low temperature (i.e., below 12 °C) seen by other workers. In the other species no such effect was apparent; K + influx decreased progressively with cooling down to 0 °C. Below 18.5 °C the apparent energy of activation ( E a ) was very low—close to that for free diffusion in water—for red cells of all species except human. Above 18.5 °C the E a was much greater and was also more variable amongst the red cells of the species chosen. Neither the inhibitors used nor cell volume changes during incubation accounted for the absence of the paradoxical effect in the species studied here. A rise in permeation of K + with cooling can, however, be produced by the addition of Ca 2+ to the medium, probably by activation of the Ca 2+-sensitive K + channel. This effect would account for previous reports of a paradoxical effect in dog and rat erythrocytes.

Involvement of (Na+ + K+)-ATPase in binding and actions of palytoxin on human erythrocytes.

Palytoxin (about 1 pM) increases the permeability of human erythrocytes. We now report its radiolabeling with 125I, followed by affinity purification on porcine kidney membranes. The resulting ligand binds fast and reversibly to intact erythrocytes. The Kd from velocity and equilibrium measurements is 2 X 10(-11) M, and the number of binding sites about 200 per cell. Binding is promoted by divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) and by borate. It is inhibited by K+ (IC50 2 mM), ouabain (IC50 3 X 10(-9) M) and ouabagenin (IC50 6 X 10(-6) M). Conversely, [3H]ouabain is displaced by the substances and concentrations mentioned, and also by palytoxin (Ki 3 X 10(-11) M). Dog erythrocytes, which are known to possess a very low (Na+ + K+)-ATPase activity, are resistant to and lack specific binding sites for palytoxin. Binding of 125I-palytoxin, like that of [3H]ouabain, depends on the state of (Na+ + K+)-ATPase. ATP depletion decreases binding of both ligands to erythrocytes. Binding of 125I-palytoxin and [3H]ouabain to red cell stroma is partially restored by ATP. In contrast to [3H]ouabain, binding of 125I-palytoxin to red cell stroma is not promoted by Mg2+ and Pi. The data show that (a) all known promoters and inhibitors of palytoxin action on human red cells do so by enhancing or decreasing its binding, (b) (Na+ + K+)-ATPase serves as a receptor for palytoxin, and (c) the antagonism by ouabain is competitive at the receptor level. They support our previous hypothesis that palytoxin increases human erythrocyte permeability by formation of pores through (Na+ + K+)-ATPase or its close vicinity.

Effects of Photoinitiators for the Visible-light Resin System on Hemolysis of Dog Erythrocytes and Lipid Peroxidation of Their Components

The visible-light (VL) polymerizing resin system with photoinitiators (photosensitizer and reducing agent) is widely used in modern dentistry. In this system, polymerization is initiated by photochemical reactions between the photosensitizers (aromatic or aliphatic ketones) and reducing agents (tertiary amines) during VL irradiation. In order to simulate the VL resin-induced toxicity in dental pulps, the present investigation was conducted to determine whether hemolysis of dog erythrocytes and lipid peroxidation of their components occur on exposure to a VL resin system containing aromatic (9-fluorenone, benzil) or aliphatic ketones (camphoroquinone) plus tertiary amine [2-(dimethylamino) ethyl methacrylate]. It was demonstrated that the hemolysis and lipid peroxidation with aromatic ketones were markedly higher than with those of aliphatic ketones, even when the latter were used at higher concentrations. It was clear that peroxidation-induced hemolysis occurred and, further, that surface-active complexes between photosensitizers and reducing agents, which are formed by irradiation, promote hemolytic activity due to their hydrophobic interactions. VL resin-induced responses to dental pulps are probably promoted due to the double effects of unpolymerized monomers and of both radicals and surface-active complexes formed during irradiation.

Evidence for a Calmodulin-Activated Ca2+ Pump ATPase in Dog Erythrocytes

Previous work in several laboratories revealed little or no Ca2+ pump ATPase activity and little or no activation of the ATPase by calmodulin (CaM) in membranes isolated from dog red blood cells (RBCs). In the present work, intact RBCs from dogs were exposed to the ionophore, A23187, in the presence of Ca2+. A rapid, apparently first order, loss of ATP occurred under these conditions. The first order rate constant was 0.0944 min-1, or approximately 47% of that found in human RBCs under the same conditions. The anti-CaM drug, trifluoperazine, inhibited the loss of ATP and the Ca2+ activation curve of ATP loss in intact cells resembled that observed for CaM-activated Ca2+ pump ATPase in isolated human membranes. Taken together, these data are consistent with the interpretation that the dog RBC membrane contains a CaM-activated Ca2+ pump ATPase.

Uric acid uptake in erythrocytes of Beagle and Dalmatian dogs

1. 1. Uric acid uptake by erythrocytes of Beagle and Dalmatian dogs has been measured, using (2- 14C) uric acid. 2. 2. Uptake was characterized by a fast and a slow component. 3. 3. Urate uptake was inhibited by certain purine and pyrimidine derivatives and by anion transport inhibitors. It was dependent on intraerythrocyte glycolysis. 4. 4. Temperature only influenced uptake by the slow component ( Q 10 = 2.6). 5. 5. Urate uptake by the slow component is apparently due to the transport into the erythrocytes by facilitated diffusion ( K m = 6.6 mmol/l, V max = 390 μmol/l/min), whereas the fast component exhibits an adsorption of urate on erythrocyte surface. 6. 6. No difference of urate uptake by erythrocytes of Beagle and Dalmatian dogs has been observed.

Involvement of [formula omitted] in binding and actions of palytoxin on human erythrocytes

Palytoxin (about 1 pM) increases the permeability of human erythrocytes. We now report its radiolabeling with 125I, followed by affinity purification on porcine kidney membranes. The resulting ligand binds fast and reversibly to intact erythrocytes. The K d from velocity and equilibrium measurements is 2 · 10 −11 M, and the number of binding sites about 200 per cell. Binding is promoted by divalent cations ( Ca 2+ > Sr 2+ > Ba 2+ ) and by borate. It is inhibited by K + (IC 50 2 mM), ouabain (IC 50 3 · 10 −9 M) and ouabagenin (IC 50 6 · 10 −6 M). Conversely, [ 3H]ouabain is displaced by the substances and concentrations mentioned, and also by palytoxin ( K i 3 · 10 −11 M). Dog erythrocytes, which are known to possess a very low ( Na + + K +)- ATPase activity, are resistant to and lack specific binding sites for palytoxin. Binding of 125I-palytoxin, like that of [ 3H]ouabain, depends on the state of ( Na + + K +)- ATPase . ATP depletion decreases binding of both ligands to erythrocytes. Binding of 125I-palytoxin and [ 3H]ouabain to red cell stroma is partially restored by ATP. In contrast to [ 3H]ouabain, binding of 125I-palytoxin to red cell stroma is not promoted by Mg 2+ and P i. The data show that (a) all known promoters and inhibitors of palytoxin action on human red cells do so by enhancing or decreasing its binding, (b) ( Na + + K +)- ATPase serves as a receptor for palytoxin, and (c) the antagonism by ouabain is competitive at the receptor level. They support our previous hypothesis that palytoxin increases human erythrocyte permeability by formation of pores through ( Na + + K +)- ATPase or its close vicinity.

Effect of onion ingestion on anti-oxidizing agents in dog erythrocytes.

The effect of onion ingestion on red cell anti-oxidizing agents such as superoxide dismutase (SOD), catalase, glucose-6-phosphate dehydrogenase (G-6-PD), glutathione peroxidase (GPx), NADH-methemoglobin reductase and reduced glutathione (GSH) was studied in relation to Heinz body hemolytic anemia in dogs. To dogs was given the onion soup equivalent to 30g of raw onion/kg body weight once a day for three days. A few days after the initiation of feeding, there was a transient decrease of SOD, catalase and G-6-PD activities to 60%, and GSH value to 70% of their predosing values with a sudden and marked Heinz body formation. These values then began to increase when Heinz bodies were observed in almost every red cells. On day 10, when red cells carrying Heinz bodies were almost cleared from blood circulation, catalase and G-6-PD reached peak values of nearly 250% of their predosing levels. Changes of GPx and NADH-methemoglobin reductase were not correlated with Heinz body counts. Methemoglobin concentration was significantly elevated and maintained in the level of 2-3% during the presence of Heinz bodies in the red cells.

New fimbrial antigen F165 from Escherichia coli serogroup O115 strains isolated from piglets with diarrhea.

Sixteen strains of Escherichia coli serogroup O115 isolated from piglets with diarrhea were examined for mannose-sensitive or mannose-resistant hemagglutination (MSHA or MRHA, respectively) for the presence of fimbriae by electron microscopy and for enterotoxigenicity by the ligated gut loop technique in 10-day-old piglets. Four strains demonstrated MRHA of sheep, goat, pig, dog, cat, chicken, and human erythrocytes but no MRHA of calf, horse, guinea pig, and rabbit erythrocytes. They were divided into pattern I (MSHA negative) and pattern II (MSHA positive). The remaining 12 strains were classified as pattern III (MRHA negative, MSHA positive) and pattern IV (hemagglutination negative). An antiserum produced against the MRHA-positive, MSHA-negative strain 4787 and absorbed by the same strain grown at 15 degrees C agglutinated all of the MRHA-positive strains but none of the MRHA-negative strains and completely inhibited the MRHA of these strains. The surface antigen against which this absorbed antiserum was directed was designated "F165." Fimbriae (pili) purified from strain 4787 hemagglutinated erythrocytes in the same mannose-resistant pattern as the strain itself and reacted with the anti-F165 antiserum in an enzyme-linked immunosorbent assay, thus demonstrating the fimbrial nature of the hemagglutinating F165 antigen. The F165 antigen showed no serological relationship with the fimbrial antigens F4, F5, F6, and "F41". A positive correlation between the presence of F165 and the lack of enterotoxigenicity was demonstrated. Thus, we found a new mannose-resistant, hemagglutinating fimbrial antigen, F165, which is produced only by nonenterotoxigenic strains of E. coli serogroup O115. The possible role of F165 as a virulence attribute of E. coli strains causing extraintestinal disease is discussed.

Inhibition of carbonic anhydrase by plasma of dogs and rabbits.

Carbonic anhydrase (CA) activity was measured by the Bowes-Davis technique in diluted hemolysates of dog erythrocytes, rabbit erythrocytes, and dog lung tissue homogenates. Plasma (from the same animal) inhibited the CA activity in each case. For 1:16,700 dilution of dog erythrocytes, the CA catalyzed the CO2 hydration reaction by 5.3 +/- 0.4-fold above the uncatalyzed rate, and half that activity was inhibited by plasma concentrations of 0.44 +/- 0.05%. Similar rabbit CA concentrations were inhibited by plasma concentrations of 1.02 +/- 0.24%. CA from dog lung tissue homogenate is only partially inhibited by plasma even at high plasma concentrations, suggesting different isozymes, at least one of which is not inhibited by plasma. The results suggest that extrapolating from artificially perfused lungs or histological observations to in vivo conditions may not be valid, and the possibility of inhibition by plasma in at least some species should be considered.

Effects of thermal trauma on dog erythrocyte ATPase and shape

Studies have been carried out on dog erythrocytes to determine the effects of heating to 57°C on membrane ATPase and erythrocyte shape. The results of these studies showed that after heating the blood there was approximately a 30 per cent decrease in membrane ATPase and an alteration of erythrocyte shape from biconcave to spherical. Additional studies of the effects of addition of arachidonic acid to erythrocyte-membrane homogenates demonstrated that this substance produced a dose-related inhibition of ATPase. If the additions were made to membranes from blood which had previously been heated, the effects of arachidonic acid and heat were additive. Addition of the hydroxyl-radical scavenger catalase to the blood before heating prevented the observed decrease in ATPase and prevented the change in shape to spherical. These results confirm the previous findings of Bessis that heat will cause a change in the erythrocyte shape from biconcave to spherical and suggest that the mechanism responsible for this change is an alteration in membrane ATPase. The observation that a similar response can be produced by arachidonic acid suggests that metabolism of this substance may generate agents which are capable of inhibiting erythrocyte membrane ATPase. The observation that catalase will prevent the ATPase inhibition and the change in erythrocyte shape suggests that hydroxyl-free radicals play an important part in this thermal trauma-induced response.

Determination of the surface tension of various species of erythrocytes by means of the solidification front technique

The solidification front technique is employed to determine the surface tension of fixed erythrocytes of dog, horse, human, chicken, and turkey. The results range from 65.5 erg/cm2 for dog erythrocytes to 67.6 erg/cm2 for turkey erythrocytes. A detailed error analysis shows that the differences obtained are statistically significant. Since cellular interactions are governed to a considerable extent by surface tension effects, it is concluded that caution needs to be exercised when results obtained for one species are used to predict the behavior of cells of another species.

Stimulation of human monocyte oxidative burst and related cytotoxicity by tumor-promoting and non-tumor-promoting diterpene esters, indole alkaloids and polyacetate-type agents.

Human peripheral blood monocytes were cultured and exposed to plant diterpenes, indole alkaloids and polyacetates with various degrees of tumor-promoting activity. The effect of the above-mentioned encounter on monocyte function was examined, as expressed by H2O2 production and lysis of dog erythrocytes by the cells, and the inhibition of 3H-PDBu binding to the monocytes by the various test agents. The most effective reagents in both activation of monocyte function and inhibition of 3H-PDBu binding were 12-0-tetradecanoyl-phorbol 13-acetate (TPA), phorbol 12, 13 dibutyrate, teleocidin, and aplysiatoxin which are known to be strong tumor promoters. Strong stimulation of monocyte function was also exerted by the weak tumor promoters, phorbol 12-retinoate 13-acetate, mezerein and debromoaplysiatoxin. Non-tumor-promoting phorbol diterpenes such as phorbol 12, 13-diacetate, phorbol 12-myristate, phorbol 13-acetate and 4-alpha TPA were 1,000 times less effective than TPA in monocyte stimulation and inhibition of 3H-PDBu binding. These results indicate that stimulation of human monocyte H2O2 production and related cytotoxicity might discriminate effectively between tumor-promoting and non-tumor-promoting reagents.

2-Chloroadenosine, a permeant for the nucleoside transporter

Human erythrocytes were shown to possess a saturable uptake mechanism for 2-chloro-adenosine (apparent K m 23 μM, 22°). Uptake by this route was inhibited by nitrobenzylthioinosine, uridine and adenosine, but adenine had no effect. In addition, uridine caused the countertransport of 2-chloroadenosine and vice versa. 2-Chloroadenosine was also shown to be an apparent competitive inhibitor of uridine influx (apparent K i value of 33 μM) and high-affinity nitrobenzylthioinosine binding (apparent K i 0.18 mM). The apparent K i value for inhibition of uridine influx was close to the apparent K m value for 2-chloroadenosine uptake. Previous studies [Jarvis et al., Biochem. J. 208, 83 (1982)] have demonstrated that dog erythrocytes do not possess a saturable transport system for uridine and adenosine. Similarly, in the present study, the entry of 2-chloroadenosine into dog erythrocytes was slow and linear with concentration. Nitrobenzylthioinosine (NBMPR) had no effect on the uptake of 2-chloroadenosine into dog erythrocytes. These results demonstrate that 2-chloroadenosine enters human erythrocytes by the same nucleoside carrier as other nucleosides. It is suggested from these data that the previous explanation that the inability of nucleoside transport inhibitors to potentiate the pharmacological effects of 2-chloroadenosine was due to the failure of the nucleoside carrier to accept 2-chloroadenosine as a permeant may have to be reassessed.

Stability of [ 3H]ouabain binding to the (Na + + K +)-ATPase solubilized with C 12E 8

The (Na + + K +)-ATPase from dog kidney and partially purified membranes from HK dog erythrocytes were labeled with [ 3H]ouabain, solubilized with C 12E 8 and analyzed by HPLC through a TSK-GEL G3000SW column in the presence of C 12E 8, Mg 2+, HPO 4 2− and glycerol at 20–23°C. The peaks of [ 3H]ouabain bound to the enzyme from dog kidney and HK dog erythrocyte membranes corresponded to each other with apparent molecular weights of 470 000–490 000. In addition, these bindings of [ 3H]ouabain to the (Na + + K +)-ATPase were observed to be stable at 20–23°C for at least 18 h after the solubilization.

Properties of an Australian isolate of bovine parvovirus type 1

An Australian bovine parvovirus isolate (BPV 267) was found to haemagglutinate human, guinea-pig, rat and dog erythrocytes, out of a range of 16 species of erythrocyte tested. The haemagglutinating activity was generally found to be both pH and temperature dependent. The virus was found to replicate best in intestinal epithelium, macrophage and lung cells, out of 9 bovine cell types tested. Highest yields of virus were obtained by the use of selected cell strains at low-passage levels which were maintained near neutral pH under conditions of high rates of cell growth. Studies of the rates of thermal inactivation with time showed the virus to be relatively stable at 37°C, 56°C and 70°C, the incorporation of serum proteins, 1 M MgCl 2 and 2 M NaCl in the medium having no influence on stability at 56°C. The virus was resistant to the action of CHCl 3, ether and 1% trypsin, and its replication was inhibited by BUDR, this effect being reversed by thymidine. Actinomycin D was found to inhibit virus replication, but only when applied during the first 8 h post-infection. Density gradient studies showed infective virus to have a density of 1.41 g cm −; haemagglutinating non-infective virus with defective morphology having a density of 1.31 g cm −3. In addition, a proportion of morphologically-complete haemagglutinating, but non-infective virus particles was found at a density of 1.36 g cm −3. The virus proved to have a mean diameter of 22 nm.

Inhibition of carbonic anhydrase in dog plasma.

Using a pH stat method, we measured the activity of carbonic anhydrase (CA) from dog erythrocytes in the presence of various amounts of dog plasma. A plasma factor appeared to be able to inhibit about 86% of the total CA activity, corresponding to the relative activity of CA II. Naiodoacetate was shown to inhibit the total CA activity up to about 13%, corresponding to the relative activity of CA I. C1- inhibited the total CA activity up to about 20%, presumably mainly through its strong influence on Ca I. It is concluded that with a degree of haemolysis of up to 3%, no appreciable plasma CA activity will occur.

Structural studies of O-glycosidic oligosaccharide units of dog erythrocyte glycophorin

Dog glycophorin, the major sialoglycoprotein of dog erythrocyte membranes, contains either N-acetyl- or N-glycolylneuraminic acid, depending upon the strain of dog. Glycolipids also contained the same sialic acid as those found in glycophorin when both materials are prepared from erythrocyte membranes of individual dogs. The O-glycosidic oligosaccharides were released from glycophorin, prepared from individual dogs, by alkaline borohydride treatment, and purified by gel filtration and ion-exchange chromatography. The structures of the reduced oligosaccharides were determined by methylation analysis and gas-liquid chromatography-mass spectrometry. The O-glycosidic oligoscharides identified were one tetrasaccharide - Neu5Ac(2→3)Gal)1→3)[Neu5Ac(2→6)[GalNAcol - two trisaccharides - Neu5Ac(2→3)Gal1→3)GaINAcol and Gal(1→3)[Neu5Ac(2→6)]GalNAcol.