Dog Erythrocytes Research Articles

Adherence activity of Porphyromonas isolated from the oral cavity of the human and dogs to epithelial cells and erythrocytes.

Adhesive activities of a strain of Porphyromonas gingivalis (previously classified as Bacteroides gingivalis) isolated from human with periodontitis, and 2 strains of Porphyromonas asaccharolyticus (previously classified as Bacteroides asaccharolyticus) from dogs with gingival inflammation to human and dog buccal epithelial cells and erythrocytes from various animal species, were compared in relation to their pathogenicities. The dog-derived P. asaccharolyticus strongly adhered to dog epithelial cells but not to human cells. The human-derived P. gingivalis, in contrast, strongly adhered to human cells but not to the dog cells. P. gingivalis strongly agglutinated all species of erythrocytes examined including human blood group erythrocytes A, B and O. On the other hand, P. asacchrolyticus and other Bacteroides were found to agglutinate rabbit and/or rat erythrocytes but not other species of erythrocytes examined. Lipopolysaccharide (LPS) from human-derived P. gingivalis weakly agglutinated rat erythrocytes. Hemagglutinating activity was not found in fimbrial material from the human derived P. gingivalis. Sonicated extract and LPS from dog-derived P. asaccharolyticus agglutinated only the rat erythrocytes. The results indicate that adherence activity of Porphyromonas was stronger to the epithelial cells of the original host animal specie for individual organism than that to the cells from other species. In contrast, there was no specific correlation between each organism and the original host animal specie in their hemagglutinating activity. It is suggested that the adherence activity of Porphyromonas to the epithelial cells determined the bacterial specie which could grow and colonize on oral surfaces in specific animals.

Postnatal hematologic development in phosphofructokinase-deficient dogs

Adult dogs with phosphofructokinase (PFK) deficiency have compensated hemolytic anemia, due to an absolute or functional deficiency of the muscle-type (M-type) subunit that normally accounts for a majority of total erythrocyte PFK activity in dogs. Potential effects of PFK deficiency on hematologic development were evaluated in dogs. Routine hematologic parameters were similar in normal and affected dogs when 1 day old, because all newborn dogs had erythrocyte PFK activities about three times that of normal adult dogs. Based on chromatographic separation of PFK isozymes and enzyme immunoprecipitation studies, the high PFK activity at birth was attributed to the predominance of the liver-type (L-type) subunit of PFK, which is negligible or absent in normal adult dog erythrocytes. Both total PFK activities and the amounts of L-type subunit present decreased dramatically during the first 6 to 8 weeks of life. The muscle-type subunit was negligible or absent at birth, but appeared and increased as the L-type decreased in normal dogs. These changes may result from the replacement of erythrocytes formed in the fetus with those formed after birth. A postnatal physiologic anemia developed to a similar degree in both affected and normal dogs because of decreases in both mean corpuscular volume and erythrocyte numbers. Reticulocyte counts were high in all dogs at birth and remained high in affected dogs, but decreased from 2 months of age onward in normal dogs. Erythrocyte 2,3-diphosphoglycerate (DPG) values were very low in all newborn pups and increased to values expected for adults in the respective groups by 2 to 4 weeks of age. A low 2,3-DPG concentration occurs in affected dogs because PFK deficiency inhibits glycolysis above the side shunt that forms 2,3-DPG.

Inherited persistence of immature type pyruvate kinase and hexokinase isozymes in dog erythrocytes.

1. Red cell pyruvate kinase (EC 2.7.1.40) and hexokinase (EC 2.7.1.1) in high and low potassium (K) dogs were shown to exist as multiple forms which were separable by electrophoresis and ion-exchange chromatography. The R2-type pyruvate kinase, which was determined to be a young type enzyme in canine red cells, was shown to be the predominant form of pyruvate kinase in high K cells. 2. The M2-type pyruvate kinase, a prototype isozyme in erythroid cells, existed in high K dog erythrocytes as well as in high K and low K dog reticulocytes. 3. Isozyme analysis of high K red cell hexokinase also showed a profile similar to that obtained for low K reticulocytes. 4. These results seem to reflect the immaturity of high K erythrocytes, which suggest that an abnormal cell differentiation or maturation may occur at an early stage of erythroid cell proliferation in high K dogs.

Palytoxin promotes potassium outflow from erythrocytes, HeLa and bovine adrenomedullary cells through its interaction with Na +, K +-ATPase

E. Habermann, M. Hudel and M.-E. Dauzenroth. Palytoxin promotes potassium outflow from erythrocytes, HeLa and bovine adrenomedullary cells through its interaction with Na +, K +-ATPase. Toxicon 27, 419–430, 1989.—Erythrocytes from four mammalian species were compared with regard to K +loss triggered by palytoxin, to Na +, K +-ATPase activity, and to ouabain sensitivity of both events. Palytoxin sensitivity ( ec 50) decreased in the order rat, man (≈ 1 pM) > cattle (≈ 500 pM) > dog (> 10 nM). Na +, K +-ATPase activity, as measured by Rb uptake, was in the series rat > man > cattle > dog. The glycoside potently inhibited both palytoxin action and ATPase activity in man, cattle and dog erythrocytes, but weakly in those from rats. Ca 2+ promoted the palytoxin effects on all erythrocytes. As shown for human erythrocytes, Sr 2+ and Ba 2+ but not Mg 2+ can substitute for Ca 2+, and sucrose can substitute for sodium chloride. Human HeLa and bovine adrenomedullary cells also lost their K + within a few min when exposed to palytoxin (1–10 pM). Ouabain acted as a palytoxin antagonist on both cell types. We conclude that: (a) the ouabain binding site of Na +, K +-ATPase is part of the palytoxin receptor in every cell type tested, (b) high palytoxin sensitivity is not necessarily accompanied by high ouabain sensitivity, and (c) active ion transport is not a precondition for the action of palytoxin or for its inhibition by ouabain.

Isolation and Preliminary Characterization of Immuno-inhibitory Factors from Dog Erythrocytes and Liver

Crude dog liver extract (DLE) inhibits proliferation of dog and human lymphocytes stimulated by phytohemagglutinin and alloantigens. While purifying this activity from dog liver, we observed that dog liver inhibitory factor (DLIF) shared properties with hemoglobin. DLIF migrated with hemoglobin during DEAE cellulose chromatography, and DLIF had oligomeric (61,900) and subunit (17,900 and 15,700) apparent molecular weights (AMW) similar to concurrently analyzed Sigma canine hemoglobin (61,100, 16,700 and 14,900). After separate cross linking, the proteins comigrated on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) gels. We therefore isolated dog erythrocyte inhibitory factor (DEIF) from red blood cells to determine if DEIF was a hemoglobin derivative. DEIF, like DLIF, separated at an isoelectric point of 5.6. DEIF also had similar subunit AMW (17,600 and 15,300) by SDS PAGE, but DEIF had much lower lymphocyte inhibitory activity (LIA = 2.27) than DLIF (LIA = 100.00). We conclude that DLIF and DEIF are similar to each other and hemoglobin, but further studies are needed to determine the function and exact structure of DLIF and DEIF.

Species-specific hemolysis of erythrocytes by T-2 toxin

The tricothecene mycotoxin, T-2 toxin interacts differently with mammalian erythrocytes. Pig, man, rabbit, guinea pig, horse, dog, rat, and mouse erythrocytes are all lysed to a varying degree by T-2 toxin. But cow, sheep, goat, buffalo, and deer erythrocytes are all resistant to hemolysis by T-2 toxin. Since erythrocytes from ruminant animals contain little or no phosphatidylcholine, perhaps the presence of phosphatidylcholine in the membrane is required for the hemolytic action of T-2 toxin. Sheep erythrocytes were used to encapsulate T-2 toxin further confirming the resistance of erythrocytes from animals with ruminant physiology to T-2 toxin lysis.

Hematological, osmotic, and scanning electron microscopic study of erythrocytes of dogs given beta-acetylphenylhydrazine.

Hematologic examinations, osmotic fragility tests, and scanning electron microscopy of erythrocytes were done on blood of dogs given 5 mg/kg of beta-acetylphenylhydrazine for 5 weeks. Reticulocytes, Heinz bodies, and serum total bilirubin values increased in the 1st week. Reticulocyte numbers peaked in the 2nd week, and reticulocytosis persisted through the 5th week. Erythrocyte, packed cell volume, and hemoglobin values decreased markedly and became lowest in the 2nd week. Mean corpuscular volume increased in the 1st week and remained increased for the duration of treatment. Erythrocyte osmotic fragility was increased after 1 week of treatment. Echinocytes were increased with a peak level of 47.6% at week 1 of treatment. Increased numbers of acanthocytes and schizocytes also were detected.

Elevated Glutathione Accelerates Oxidative Damage to Erythrocytes Produced by Aromatic Disulfide

It has been shown that certain dogs have erythrocytes characterized by an inherited high concentration of reduced glutathione (GSH), five to seven times the normal level (high-GSH RBCs). We examined whether increased GSH in dog erythrocytes leads to increased protection against oxidative damage induced by acetylphenylhydrazine (APH) and/or 4-aminophenyl disulfide (4-AD). When erythrocytes were incubated with 30 mmol/L APH, the Heinz body count was appreciably higher in normal RBCs than in high-GSH RBCs, while there was no difference in the increase of the methemoglobin (metHb) concentration in both RBCs. In contrast, both the Heinz body count and metHb production were much higher in high-GSH RBCs than in normal RBCs when erythrocytes were incubated with 4-AD. Furthermore, the generation of the superoxide in erythrocytes treated with 4-AD, which was measured by spin trapping combined with electron spin resonance (ESR), was obviously higher in high-GSH RBCs than in normal RBCs. These results clearly indicate that erythrocyte GSH is an important defense against oxidative damage induced by certain compounds such as APH, but that, in contrast, elevated GSH appears to accelerate oxidative damage to erythrocytes produced by aromatic disulfides, such as 4-AD, which generated a superoxide in erythrocytes via its redox reaction with GSH.

Elevated glutathione accelerates oxidative damage to erythrocytes produced by aromatic disulfide

It has been shown that certain dogs have erythrocytes characterized by an inherited high concentration of reduced glutathione (GSH), five to seven times the normal level (high-GSH RBCs). We examined whether increased GSH in dog erythrocytes leads to increased protection against oxidative damage induced by acetylphenylhydrazine (APH) and/or 4- aminophenyl disulfide (4-AD). When erythrocytes were incubated with 30 mmol/L APH, the Heinz body count was appreciably higher in normal RBCs than in high-GSH RBCs, while there was no difference in the increase of the methemoglobin (metHb) concentration in both RBCs. In contrast, both the Heinz body count and metHb production were much higher in high-GSH RBCs than in normal RBCs when erythrocytes were incubated with 4-AD. Furthermore, the generation of the superoxide in erythrocytes treated with 4-AD, which was measured by spin trapping combined with electron spin resonance (ESR), was obviously higher in high-GSH RBCs than in normal RBCs. These results clearly indicate that erythrocyte GSH is an important defense against oxidative damage induced by certain compounds such as APH, but that, in contrast, elevated GSH appears to accelerate oxidative damage to erythrocytes produced by aromatic disulfides, such as 4-AD, which generated a superoxide in erythrocytes via its redox reaction with GSH.

Haemagglutinating activity of Campylobacter pylori

Thirty-one isolates of Campylobacter pylori, screened for their ability to agglutinate a panel of erythrocyte species, could be divided into two phenotypic groups on the basis of their ability to agglutinate human A and O erythrocytes, a property which correlated strongly with their ability to agglutinate horse and cat erythrocytes. Isolates which agglutinated human red blood cells exhibited a broad-spectrum haemagglutination profile on other red blood cells including dog, goat, guinea-pig, ox, rat and sheep erythrocytes. Agglutination dog, guinea-pig, horse and human erythrocytes by C. pylori was mannose-resistant. Haemagglutination was not inhibited by other saccharides tested nor by two glycoproteins or serine. The bacterial ligand was protease- and heat-sensitive. Neither protease nor neuraminidase treatment of erythrocytes prevented agglutination.

Haemagglutinating activity of Campylobacter pylori

Thirty-one isolates of Campylobacter pylori, screened for their ability to agglutinate a panel of erythrocyte species, could be divided into two phenotypic groups on the basis of their ability to agglutinate human A and O erythrocytes, a property which correlated strongly with their ability to agglutinate horse and cat erythrocytes. Isolates which agglutinated human red blood cells exhibited a broad-spectrum haemagglutination profile on other red blood cells including dog, goat, guinea-pig, ox, rat and sheep erythrocytes. Agglutination dog, guinea-pig, horse and human erythrocytes by C. pylori was mannose-resistant. Haemagglutination was not inhibited by other saccharides tested nor by two glycoproteins or serine. The bacterial ligand was protease- and heat-sensitive. Neither protease nor neuraminidase treatment of erythrocytes prevented agglutination.

Column chromatographic separation of cells using aqueous polymeric two-phase systems

Cell separation using aqueous polymeric two-phase systems is well established. For separations of cells having similar partition coefficients a multistep countercurrent distribution procedure has to be used. However, its operation is limited by time and apparatus constraints. As an alternative strategy we have developed a chromatographic technique in which the dextran-rich phase of a dextran/polyethylene glycol (PEG) phase system is immobilized onto derivatized agarose beads. The PEG-rich phase is used as the eluent. Inclusion of PEG-fatty acid affinity ligand gradients into the eluent produces separations of mammalian erythrocytes based on the differential interaction between the fatty acid and the erythrocyte membranes. A model separation of dog and human erythrocytes has been carried out.

Hemagglutinating properties of Actinobacillus pleuropneumoniae.

A total of 26 isolates of Actinobacillus pleuropneumoniae were tested for their ability to agglutinate erythrocytes of different origins. Seven different hemagglutination patterns were found. Ten (38%) isolates did not agglutinate any of the erythrocytes tested. The remaining 16 (62%) isolates agglutinated human erythrocytes, and among these, 12 also agglutinated rat, cat, dog, guinea pig, or bovine erythrocytes. No correlation was found between the seven different hemagglutination patterns observed and the serotypes. Hemagglutination activity was destroyed by heating at 100 degrees C as well as by formaldehyde treatment, but was not affected by heating at 60 degrees C, by treatment with trypsin or pronase, or by homogenization of bacterial cells. No fimbriae were observed on examination of bacterial cells negatively stained with phosphotungstate using electron microscopy. Hydrophobic surface properties of the isolates were evaluated. All the isolates appear to possess a hydrophilic cell surface. The present study provides evidence that certain isolates of A. pleuropneumoniae possess hemagglutinating properties which do not appear to be mediated by fimbriae or to involve hydrophobic interactions.

Identification, purification, and characterization of a stilbenedisulfonate binding glycoprotein from canine kidney brush border membranes. A candidate for a renal anion exchanger.

Canine renal brush border membrane proteins that bind stilbenedisulfonate inhibitors of anion exchange were identified by affinity chromatography. A 130-kDa integral membrane glycoprotein from brush border membrane was shown to bind specifically to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate immobilized on Affi-Gel 102 resin. The bound protein could be eluted effectively with 1 mM 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS). The 130-kDa protein did not bind to the affinity resin in the presence of 1 mM BADS or when the solubilized extract was covalently labeled with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). This protein was labeled with [3H]H2DIDS, and the labeling was prevented by BADS. The 130-kDa protein did not cross-react with antibody raised against human or dog erythrocyte Band 3 protein. The 130-kDa protein was accessible to proteinase K and chymotrypsin digestion in vesicles but not to trypsin. The 130-kDa protein was sensitive to endo-beta-N-acetylglucosaminidase F treatment both in the solubilized state and in brush border membrane vesicles showing that it was a glycoprotein and that the carbohydrate was on the exterior of the vesicles. This glycoprotein was resistant to endo-beta-N-acetylglucosaminidase H treatment suggesting a complex-type carbohydrate structure. The protein bound concanavalin A, wheat germ agglutinin, and Ricinus communis lectins, and it could be purified using wheat germ agglutinin-agarose.

Isolation and characterisation of dog uropathogenic Escherichia coli strains and their fimbriae.

A number of Escherichia coli strains have been isolated from dogs with urinary tract infections. These strains have been characterised with respect to their O, K, H, and fimbrial antigens, colicin production, antibiotic resistance, plasmid content and their ability to haemagglutinate erythrocytes from various species. Crossed immunoelectrophoresis of fimbrial extracts, as well as the reaction of partly purified fimbriae of a number of these strains with monoclonal antibodies revealed homology or a strong crossreaction with an F12 fimbrial subunit protein of human uropathogenic E. coli strains. Unlike human F12 fimbriae producing strains, the dog isolates did agglutinate dog erythrocytes in the presence of D-mannose but not human erythrocytes, indicating that the adhesin carried by these strains is different from the adhesin on fimbriae of human uropathogenic E. coli. Similar indications were obtained from experiments with latex beads coated with the receptor for P-fimbriae. These beads were agglutinated by Escherichia coli strains from human urinary tract infections, but not by the dog isolates described here. Preliminary adhesion experiments of human and dog Escherichia coli to human bladder epithelial and canine kidney epithelial cells also showed differences in adhesion depending on the origin of the strain tested.

A dog possessing high glutathione (GSH) and K concentrations with an increased Na, K-ATPase activity in its erythrocytes.

We have studied a female mongrel dog found in Kanagawa Prefecture, Japan. This dog was selected and examined thoroughly because she naturally maintained a high glutathione (GSH) concentration in her erythrocytes and did not exhibit any clinical signs or hematologic disorders. Erythrocytes from this animal demonstrated high K and low Na concentrations, as well as accumulation of the amino acids, glutamic acid, aspartic acid and glutamine. The Na, K-ATPase activity was also markedly elevated and the osmotic fragility of the dog's erythrocytes was found to be significantly increased. Crossbreeding of our dog with a normal dog and also with a heterozygous carrier dog revealed that the genetic abnormality possessed by our dog is transmitted as an autosomal recessive trait. All of the clinical data obtained from studying this animal strongly suggest that it possesses a genetic trait similar to that of the HK dogs previously described by Maede.

The metabolism of aminophenols in erythrocytes.

1. Like 2- and 4-dimethylaminophenol, 4-aminophenol in the presence of oxyhaemoglobin forms numerous adducts with glutathione (GSH). Using 14C-4-aminophenol and 3H-glutathione, ten different thioethers were isolated, by h.p.l.c., with isotope ratios of 1:1, 1:2, 1:3, respectively. The structural identification of the different thioethers is under current investigation. 2. In erythrocytes of human and dog, and in dog blood, in vivo, the same pattern of 4-aminophenol conjugates with GSH was found. 3. In vivo, 5% of administered 4-aminophenol is converted into thioethers within erythrocytes, accompanied by a 60% decrease in the cellular GSH, indicating the role of erythrocytes in the biotransformation of xenobiotics.

Column-based separation of erythrocytes using aqueous polymeric two-phase systems

Cell separation using aqueous polymeric two-phase systems is well established. For separations of cells having similar partition coefficients a multi-step counter-current distribution procedure has been used. As an alternative strategy, we have developed a column-based technique in which the dextran-rich phase of a dextran-polyethylene glycol (PEG) phase system is immobilized onto derivatized chromatography beads. The PEG-rich phase is used as the eluent. A separation of erythrocytes from different species utilizing an affinity ligand was performed. The following behaviour was observed: (i) no elution occurs below a critical affinity ligand concentration; (ii) cell recovery is proportional to affinity ligand concentration; (iii) elution volume is independent of affinity ligand concentration. The above suggest that no multi-step partitioning occurs. However, the separation achieved is considerably better than in a corresponding single-tube separation. A 1:1 mixture of dog and human erythrocytes was separated to a purity of > 98% using the column-based technique. The corresponding optimized single-tube separation gave a maximum purity of 84%.

Dog erythrocyte rosette-forming lymphocyte: blockage by OKT11 monoclonal antibody.

Human peripheral blood mononuclear cells (PBM) were separated into sheep erythrocyte rosette-forming (Es+) and non Es+ cells by the Ficoll-Hypaque gradient sedimentation method. Thirty-eight percent of the Es+ cells formed rosettes with dog erythrocytes and were designated as Es+Ed+ cells. The remaining Es+ cells were designated as Es+Ed- cells. Only a few non Es+ cells formed rosettes with dog erythrocytes. Among Es+Ed+ cells, T4 antigen-positive cells were observed approximately 1.7 times as often as T8 antigen-positive cells, when measured by staining with OKT4 or OKT8 monoclonal antibody. Among Es+Ed- cells, however, T4 and T8 antigen-positive cells were observed in almost equal proportion. Preincubation of PBM with OKT11 monoclonal antibody, but not with OKT4 monoclonal antibody, inhibited the rosette formation with dog as well as sheep erythrocytes. These results indicated that Es+Ed+ cells were a subpopulation of T-cells in which a majority of the cells were T4 antigen-positive, and that the binding sites of dog erythrocytes on human T-cells was closely linked with that of sheep erythrocytes.

Some properties of flammutoxin from the edible mushroom Flammulina velutipes

A. W. Bernheimer and J. D. Oppenheim. Some properties of flammutoxin from the edible mushroom Flammulina velutipes. Toxicon 25, 1145 – 1152, 1987. — A cytolytic toxin from the basidiocarps of the edible mushroom Flammulina velutipes was purified to homogeneity. The lysin, flammutoxin, is a single polypeptide chain of M r , 32,000 and p K about 5.4. It contains unusually large amounts of tryptophan, serine and glycine, and few or none of the sulfurcontaining amino acids. Erythrocytes of rat, rabbit, guinea pig, man, mouse, cat and dog were sensitive to lysis, in that order, whereas erythrocytes of sheep, ox, goat, swine and horse were largely or completely resistant to lysis. The toxin appears not to be a phospholipase and it was not inhibitable by any of a variety of lipids. Hemolysis probably involves alteration of the erythrocyte membrane, with formation of submicroscopic ion channels, and it appears to be of the osmotic type. In some respects flammutoxin resembles phallolysin, a cytolytic toxin obtained from the mushroom Amanita phalloides.