Dog Erythrocytes Research Articles

Flow cytometric assessment of canine erythrocytes and platelets for dog erythrocyte antigen 1.1

In human medicine, transfusion of ABO-mismatched platelets has been associated with shortened platelet survival and refractoriness to platelet transfusion because of expression of certain blood group antigens on platelets. It remains unknown if canine platelets express dog erythrocyte antigens (DEAs). The aim of this study was to develop a flow cytometric assay for DEA 1.1 and determine whether DEA 1.1 is present on canine platelets. Blood was collected from 172 clinically healthy dogs. Platelets and erythrocytes from each dog were tested for DEA 1.1 by flow cytometry using anti-DEA 1.1 blood-typing sera. Erythrocytes from each dog were also assessed for DEA 1.1 using a standard tube-typing test (T1) and using a second tube method (T2), if the flow cytometric and T1 results differed. Using flow cytometry, DEA 1.1 was detected on erythrocytes of all 110 dogs shown by T1 or T2 testing to be DEA 1.1-positive. Initial results of the T1 test had a diagnostic accuracy of 93% (160 correct/172 tests). The frequency of erythrocyte DEA 1.1 positivity in previously untyped dogs (n = 118) was 56%. DEA 1.1 expression was not detected on platelets from DEA 1.1-positive dogs. Flow cytometry was a reliable method for detection of DEA 1.1 on canine erythrocytes. The absence of DEA 1.1 on platelets from DEA 1.1-positive dogs suggests that their platelets do not express DEA 1.1 and will not induce production of anti-DEA 1.1 antibodies that might lead to platelet refractoriness or reactions to a subsequent transfusion of DEA 1.1-positive erythrocytes.

Fatty acid patterns of dog erythrocyte membranes after feeding of a fish-oil based DHA-rich supplement with a base diet low in n-3 fatty acids versus a diet containing added n-3 fatty acids

BackgroundIn dogs, increasing the tissue n-3 fatty acid (FA) content is associated with potential benefit in some medical conditions, e.g. atopic dermatitis, cancer or heart disease. Therefore effectively and conveniently increasing tissue n-3 FA levels in dogs is of interest. Incorporation of dietary n-3 FA into cell membranes may be studied by FA analysis of erythrocyte membranes (EM), because of the correlation of its FA composition with the FA composition of other cells. Aim of the study was to determine whether an n-3 FA additive added to a control diet is as effective in increasing EM n-3 FA content as feeding an n-3 FA enriched diet. Furthermore the time course of the incorporation of dietary n-3 FA into canine EM was investigated.MethodsThirty dogs were randomly divided into three dietary groups with ten dogs per group. CONT got a dry dog food diet which did not contain EPA or DHA. FO got a dry dog food diet with a high EPA and DHA content. ADD got the CONT diet combined with an n-3 FA additive rich in DHA and EPA. After a feeding period of 12 weeks the additive was discontinued in ADD and these dogs were fed CONT diet for another four weeks to observe washout effects. Erythrocyte lipids were extracted from venous blood samples and their FA composition was determined by gas chromatography. The Mann-Whitney-U-test was used to detect significant differences between the different groups and time points.ResultsAfter one week the proportions of n-3 FA, DHA and EPA were already significantly increased in ADD and FO, apparently reaching a plateau within eight weeks. In our study DHA and not EPA was preferably incorporated into the EM. After discontinuing the administration of the additive in ADD, the n-3 FA values declined slowly without reaching baseline levels within four weeks.ConclusionsIn dogs, an increase of dietary n-3 FA content leads to a rapid inclusion of n-3 FA into EM, regardless of whether the n-3 FA are offered as an enriched diet or as a normal diet supplemented with an n-3 FA additive.

Invasion of canine erythrocytes by Bartonella vinsonii subsp. berkhoffii

Bartonella vinsonii subsp. berkhoffii is a recognized cause of endocarditis in dogs and human patients and has been associated with cardiac arrhythmias, myocarditis, granulomatous lymphadenitis, polyarthritis, and granulomatous rhinitis in dogs. Little is known regarding the mode of transmission or cellular localization of this bacteria following infection of a canine host. The aim of the current study was to determine whether erythrocytes may serve as a site of infection by B. vinsonii subsp. berkhoffii. In the study, we successfully demonstrate the invasion of canine erythrocytes by a B. vinsonii subsp. berkhoffii genotype III strain using an in vitro model system. Dog erythrocytes were incubated with B. vinsonii subsp. berkhoffii after which tubes were treated with gentamicin at 12, 24, and 48h post-inoculation. After gentamicin elimination of extracellular bacteria, there was a gradual increase in intra-erythrocytic bacteria, as assessed by colony forming units per ml, at each collection time point. The largest recovery of intracellular bacteria occurred at 48h post-infection. These results suggest that canine erythrocytes may serve in the maintenance of bacteremia due to B. vinsonii subsp. berkhoffii within an infected host.

Prevalence of dog erythrocyte antigen 1.1 in dogs in Switzerland evaluated with the gel column technique

Canine blood typing has become an established and essential laboratory test due to the rising demand for safe and efficient blood transfusions. The most immunogenic and clinically important blood type is DEA 1.1. Little is known about DEA 1.1 frequencies or special characteristics among different canine breeds. 304 dogs were tested for DEA 1.1. DEA 1.1-typing was performed using a commercial gel column technique (ID-Gel Test Canine DEA 1.1, DiaMed, Cressier, Switzerland). Fifty-three percent of all tested dogs reacted positive for DEA 1.1, whereas 49 % of the mixed breeds tested DEA 1.1-positive. All Bernese mountain dogs (n = 22) and Rottweilers (n = 9) tested positive for DEA 1.1, while all Boxers (n = 8), Flat-Coated Retrievers (n = 9), and Border Collies (6) tested negative for DEA 1.1. The prevalence of DEA 1.1 in dogs in Switzerland was found to be comparable to that reported from other countries. The tested breeds were found to differ considerably in the frequency of DEA 1.1. This knowledge is useful for selection of blood donors. However, DEA 1.1 blood typing of donor and recipient prior to transfusion and cross matching in sensitized dogs is unavoidable.

Frequency of dog erythrocyte antigen 1.1 expression in dogs from Portugal

Dog erythrocyte antigen (DEA) 1.1 is the antigen considered most responsible for severe hemolysis owing to incompatible blood transfusions in previously sensitized dogs. Few reports describe the frequency of DEA 1.1 expression in European dogs, and there are no reports in dogs from Portugal. The aims of this study were to identify the frequency of DEA 1.1 expression in Portuguese dogs, to examine the relationship between phenotypic traits and expression of this blood group, and to assess the risk of transfusing blood that is not typed or cross-matched. Expression of DEA 1.1 was determined in 274 dogs using a migration gel test. Weight, sex, breed, and hair length and color were recorded for each dog. Results were analyzed by descriptive statistical analysis, probabilistic analysis, and χ(2)-tests. Of 274 dogs, 56.9% were DEA 1.1-positive and 43.1% were DEA 1.1-negative. All Boxers, German Shepherds, and Dobermans were DEA 1.1-negative, whereas all Saint Bernards, 88.9% of Golden Retrievers, 88.2% of Rottweilers, and 61.4% of mixed breed dogs were DEA 1.1-positive. A significant relationship between DEA 1.1 expression and phenotypic traits was not found. The probability of sensitization of recipient dogs following first-time transfusion with blood that was not typed or cross-matched was 24.5%; the probability of an acute hemolytic reaction following a second transfusion with blood from any other donor in the absence of pretransfusion compatibility testing was 6%. The frequency of DEA 1.1 expression in dogs in Portugal is high, and there is a potential risk of sensitization following transfusion with blood that is not typed or cross-matched. Breed-related frequencies may help predict DEA 1.1-positivity, but the best practice is to type and cross-match blood before transfusion.

Evaluation and evidence of natural gangliosides with two unsaturated bonds in the ceramide structure obtained by a combination of MALDI-MS and NMR spectroscopy

Gangliosides are membrane-associated glycosphingolipids. N-Acetyl GM3 and N-glycolyl GM3 are two tumor-associated antigens expressed in cancer tissues such as melanoma and mammalian cancer. In order to use these antigens in GM3-based vaccines for patients with early stage cancer, the synthetic version is recommended to avoid the risk of animal virus transmission from the source. However, the isolation of natural gangliosides is of comparative value for the structural characterization. The structures of N-acetyl and N-glycolyl GM3 extracted from dog and horse erythrocytes were evaluated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nuclear magnetic resonance techniques; additionally, the natural N-acetyl ganglioside was compared to a synthetic one. In addition to the main compound with C24:0 fatty acid chain, a minor component with an additional unsaturation in the ceramide chain was detected, in both the dog and the horse gangliosides. This paper shows spectroscopic evidence of the aforementioned compounds.

Isolation and Characterization of a Novel Cardiac Stimulatory and Haemolytic Protein from the Sea Anemone Urticina piscivora (Sebens and Laakso)

Ethanolic extraction of the whole bodies of the sea anemone Urticina piscivora, collected from the west coast of Canada contained a protein UpI with potent cardiac stimulatory and haemolytic activities. The protein was purified by gel chromatography, cation exchange, preparative and analytical reverse phase HPLC and shown to have a molecular size of approximately 28 kDa and pI of >9·3. The partial amino acid sequence as determined by automated Edman degradation of whole UpI is D1E-NEN5LYGPN10ENKAK15AKDLT20A GASY25LTKEA30GCTKL35QAGCT40MYQAY45N. The sequence was not identical to any of the known cardiotonic polypeptides and proteins, or cytolysins, already characterized from sea anemones. However it showed some sequence similarity to the phospholipase A2-like bungarotoxins from the snake venom of Bungarus multicintus. Even though U. piscivora belongs to the family Actiniidae, UpI lacks the initial N-terminal residues common to sea anemone proteins from this family. Using rat left atrial tissues, UpI had an ED50 of 8·1 × 10−9 M. We have shown UpI to be a potent haemolysin on erythrocytes of rat, guinea-pig, dog, pig and human. Like most sea anemone cytolysins, the haemolytic action is inhibited in a dose dependent manner by the phosholipid sphingomyelin. It was also ichthyotoxic. We thus conclude, that UpI is a novel cardiac stimulant and cytolysin by virtue of its biological action, molecular size and biochemical data. We believe we have successfully characterized for the first time a member of the third class of sea anemone cytolysin described as having phospholipase A2-like action, but for which no biochemical data are yet available.

Japanese Shiba dogs possessing erythrocytes with high Glut-1 activity and high ascorbic acid recycling capacity

Glut-1 was discovered in erythrocytes of some Japanese Shiba dogs. They have Glut-1 as well as Glut-4 in erythrocytes, while other Shiba dogs and other breeds have only Glut-4. Dog erythrocytes with Glut-1 showed much higher affinity for entry of glucose and oxidized ascorbic acid and greater capacity for ascorbic acid (AA) recycling than those with only Glut-4. Dog is among numerous AA-synthesizing species that normally do not have Glut-1 in erythrocytes. However, this variant of dog is the first example of an AA-synthesizing animal which maintains Glut-1 in erythrocytes throughout life. Shiba dogs can be a new animal model for further understanding the mechanism of regulation and functions of AA in the organism.

331 BLOOD ANTIGEN-COMPATIBILITY BETWEEN CLONED BEAGLES AND TRANSGENIC CLONED BEAGLES

It is known that cloned animals produced by somatic cell nuclear transfer (SCNT) using the same donor cells show immunological compatibility for tissue transplantation. However, immunological compatibility for tissue transplantation or blood transfusion between cloned animals and transgenic cloned animals originating from the same donor cell has not been assessed until now. The objective of this study was to evaluate the compatibility of blood group antigens in cloned dogs with each other, in cloned dogs and transgenic cloned dogs, and in transgenic cloned dogs with each other by a crossmatching test. In Lee’s group, 2 cloned beagles (BG1, 2) were produced from fetal fibroblasts (BF3) using SCNT. Serially, 4 transgenic cloned beagles (Ruppy 1–3, 5) were also genetically engineered from the same donor cell, BF3, although with the transfection of the red fluorescent protein (RFP) gene. All 6 beagles shared the same genetic background except for RFP gene insertion in their genome. Canine blood antigen is labelled as dog erythrocyte antigen (DEA), a critical factor for determining blood antigen compatibility during a crossmatching test. Blood samples were collected from all tested dogs. Serum and red blood cells (RBC) were separated; RBC of all dogs were washed 3 times with 0.9% saline, and a 4% RBC suspension was made from the washed cells. They were mixed in the following experimental designs. In experiment 1, an RBC suspension of cloned beagles was combined with equal volumes of another cloned beagle’s serum. In experiment 2, an RBC suspension of Ruppy 1–3, 5 was mixed with an equal volume of another transgenic cloned beagle’s serum; the reverse reaction was also performed. In experiment 3, an RBC suspension of cloned beagles was mixed with the serum of transgenic cloned beagles and the reverse reaction was performed. All the mixtures were incubated at 37°C for 20 min, centrifuged, and then assessed for hemolysis or agglutination. In experiments 1 and 2, no samples showed any evidence of hemolysis. However, in contrast to the other experiments, experiment 3 showed a different pattern. Although the RBC of transgenic cloned dogs showed hemolysis when mixed with the serum of cloned dogs, the RBC of cloned dogs did not react with the serum of transgenic cloned dogs. From the results, we see that the 4 transgenic cloned beagles could not be donors for blood transfusion, but the 2 cloned dogs could be universal donors for all. In conclusion, cloned beagles and transgenic cloned beagles show blood antigen compatibility within themselves. However, the 4 transgenic cloned beagles showed blood antigen incompatibility with the 2 cloned beagles. This study was supported by Korean MEST, through NRF (grant #M10625030005-10N250300510), and BK21 program, SNU foundation (Benefactor; RNL BIO) and Natural Balance Korea.

Soluble parasite antigens from Babesia canis do not directly activate the kallikrein system in dogs infected with Babesia canis

Soluble parasite antigens (SPA) from Babesia canis have been shown to induce protective immunity when used as vaccine. In order to explain the immune mechanisms of vaccination, the precise role of SPA in the pathogenesis of canine babesiosis is under investigation. Earlier studies suggested that the plasma kallikrein system is central in the pathogenesis of babesiosis, malaria and trypanosomosis, and significant plasma kallikrein activation during acute B. bovis and P. knowlesi infections has been described. In the studies presented here dogs were experimentally infected with B. canis to investigate whether the plasma kallikrein system is activated during babesiosis infection. Results showed that prekallikrein levels decreased during episodes of peak parasitaemia. No effect was found on the kallikrein levels. In order to determine whether B. canis SPA could activate plasma kallikrein, dogs were infused with variable amounts of B. canis SPA and plasma samples were taken for (pre-) kallikrein determination. The results indicated that B. canis SPA did not affect plasma (pre-) kallikrein levels. In addition, the effect of B. canis SPA on (pre-) kallikrein levels in normal dog plasma was determined in vitro. Again, no effect on (pre-) kallikrein levels was found. The results suggest that, although the kallikrein pathway may be involved in B. canis-associated pathology, the system is not directly activated by B. canis SPA. Furthermore, infusion of B. canis SPA as well as stroma of normal dog erythrocytes triggered the production of the acute phase reactant, C-reactive protein. This suggests that the inflammatory response that is triggered during B. canis infection could be in part due to the release and exposure of self molecules. The implications of these findings are discussed.

Prevalence of dog erythrocyte antigens in retired racing Greyhounds

Blood groups in dogs are designated as dog erythrocyte antigen (DEA) 1.1, 1.2, 3, 4, 5, 7, and Dal. There is limited information about the frequency of different antigens in Greyhound dogs, despite their frequent use as blood donors. The aims of this study were to determine the frequencies of DEA 1.1, 1.2, 3, 4, 5, and 7 in Greyhounds, to compare the frequencies with those of non-Greyhound dogs, and to evaluate the presence of naturally occurring anti-DEA antibodies. Blood was collected from 206 Greyhound and 66 non-Greyhound dogs being screened as potential blood donors. Blood-typing was performed at Animal Blood Resources International by tube agglutination utilizing polyclonal anti-DEA antibodies. Of the Greyhound dogs, 27/206 (13.1%) were positive for DEA 1.1, and this frequency was significantly lower (P<.0001) than for non-Greyhound dogs of which 40/66 (60.6%) were DEA 1.1-positive. The frequency of positivity for both DEA 1.1 and 1.2 was also lower in Greyhounds (P<.0001). There were no significant differences between Greyhounds and non-Greyhounds for DEA 1.2, 3, 4, 5, or 7. All 137 dogs (113 Greyhounds and 24 non-Greyhounds) that were evaluated for naturally occurring anti-DEA antibodies in serum were negative. A higher percentage of Greyhound dogs (57.3%, 118/206) were considered "universal donors" (negative for all DEAs except DEA 4) compared with non-Greyhound dogs (28%, 13/46). The frequency of positivity for DEA 1.1 in our population of Greyhounds was significantly lower than previously reported for dogs. Furthermore, a large majority of Greyhounds met the criteria for universal donors.

ORIGINAL RESEARCH: Dog erythrocyte antigens 1.1, 1.2, 3, 4, 7, and Dal blood typing and cross-matching by gel column technique

Testing for canine blood types other than dog erythrocyte antigen 1.1 (DEA 1.1) is controversial and complicated by reagent availability and methodology. The objectives of this study were to use available gel column technology to develop an extended blood-typing method using polyclonal reagents for DEA 1.1, 1.2, 3, 4, 7, and Dal and to assess the use of gel columns for cross-matching. Dogs (43-75) were typed for DEA 1.1, 1.2, 3, 4, 7, and Dal. METHODS included tube agglutination (Tube) using polyclonal reagents, a commercially available DEA 1.1 gel column test kit (Standard-Gel) using monoclonal reagent, and multiple gel columns (Extended-Gel) using polyclonal reagents. Blood from 10 recipient and 15 donor dogs was typed as described above and cross-matched using the gel column technique. Of 43 dogs typed for DEA 1.1, 23, 25, and 20 dogs were positive using Standard-Gel, Extended-Gel, and Tube, respectively. Typing for DEA 1.2 was not achievable with Extended-Gel. For 75 dogs typed for DEA 3, 4, and 7, concordance of Extended-Gel with Tube was 94.7%, 100%, and 84%, respectively. Dal, determined only by Extended-Gel, was positive for all dogs. Post-transfusion major cross-matches were incompatible in 10 of 14 pairings, but none were associated with demonstrable blood type incompatibilities. Gel column methodology can be adapted for use with polyclonal reagents for detecting DEA 1.1, 3, 4, 7, and Dal. Agglutination reactions are similar between Extended-Gel and Tube, but are more easily interpreted with Extended-Gel. When using gel columns for cross-matching, incompatible blood cross-matches can be detected following sensitization by transfusion, although in this study incompatibilities associated with any tested DEA or Dal antigens were not found.

Parallel Reductions in Stomatin and Na,K-ATPase through the Exosomal Pathway during Reticulocyte Maturation in Dogs: Stomatin as a Genotypic and Phenotypic Marker of High K+ and Low K+ Red Cells

Dogs can be divided into two genetic groups (a minor HK phenotype and a major LK phenotype) based on erythrocyte monovalent cation concentrations, which are controlled by the putative hk and lk allelic genes. HK dogs retain Na,K-ATPase in their erythrocytes due to the high activity of the enzyme in their precursor cells, whereas total loss of reticulocyte Na,K-ATPase occurs in LK dogs. Here, we report that the levels of the lipid raft-associated membrane protein stomatin decrease in parallel with those of Na,K-ATPase during reticulocyte maturation due to its extrusion in exosomes. The stomatin content of HK reticulocytes is higher than that of LK reticulocytes, and remains in the erythrocytes at levels compatible with that in human erythrocytes. However, it is almost absent from LK erythrocytes with the lk/lk genotype; similar to the deficiency seen in human red cells with overhydrated stomatocytosis. LK erythrocytes from hk/lk genotype dogs show reduced, but not negligible, levels of stomatin. These results indicate that the erythrocyte stomatin level is a suitable genotypic marker for the HK/LK red cell phenotype, and suggests a functional association between stomatin and Na,K-ATPase. The absence of morphological abnormalities in the erythrocytes of stomatin-deficient LK dogs also confirms that stomatin deficiency and stomatocytic shape change are independent from each other.

The role of para‐aminophenol in acetaminophen‐induced methemoglobinemia in dogs and cats

Acetaminophen (APAP) overdose in most species is associated with hepatotoxicity because of the metabolite N-acetyl-p-benzoquinoneimine (NAPQI). In dogs and cats, APAP overdose primarily causes methemoglobinemia and hemolysis. Although NAPQI has been proposed as the responsible intermediate in dogs and cats, it lacks chemical or pharmacokinetic characteristics that favor methemoglobin formation. We hypothesized that para-aminophenol (PAP) rather than NAPQI induces methemoglobinemia and that deficient arylamine N-acetyltransferase (NAT) activity in dogs and cats contributes to this species-dependent methemoglobinemia. Erythrocytes from dogs, cats, mice, and rats were exposed in vitro to APAP, NAPQI, and PAP. Only PAP induced methemoglobin and it induced more methemoglobin formation in dog and cat than rat and mouse erythrocytes. PAP also induced more methemoglobin in erythrocytes from Nat1/Nat2 knockout mice than wildtype (WT) mouse erythrocytes (P < 0.05), but less than in dog and cat erythrocytes (P < 0.01). APAP and PAP toxicity were compared in vivo in WT and Nat1/Nat2 knockout mice. APAP caused no hematotoxicity while PAP induced more methemoglobin in NAT1/NAT2 knockout mice than in WT mice (P < 0.05). These results support the hypothesis that PAP is the metabolite responsible for APAP-induced methemoglobinemia and that deficient NAT activity in dogs and cats contributes to this species-dependent toxicity.

Lack of Evidence of Pregnancy‐Induced Alloantibodies in Dogs

It is controversial whether or not pregnant bitches become sensitized to red blood cell (RBC) antigens. Bitches do not develop alloantibodies to RBC antigens during gestation and can be used safely as blood donors. The study group included 35 healthy female dogs with a prior history of 1 (n = 12), 2 (n = 14), or >or= 3 (n = 9) pregnancies. The control group consisted of 15 healthy female dogs without any history of pregnancy. All dogs were blood typed for dog erythrocyte antigens (DEA) 1.1, 1.2, 3, 4, 5, and 7 using ethylenediaminetetraacetic acid blood samples and polyclonal antisera. Antibody screening was performed with serum and canine RBC panels of known blood type. An autocontrol and direct antiglobulin test were performed to rule out the presence of autoantibodies. The only alloantibodies identified were those against DEA 7 and the prevalence of anti-DEA 7 alloantibodies was similar in dogs with known history of pregnancy (11.4%) and in the control group (13.3%). These results confirm previous studies and clinical transfusion medicine experience. Naturally occurring anti-DEA 7 alloantibodies have been reported but their clinical relevance has not been shown. Pregnancy does not appear to sensitize dogs to RBC antigens. Consequently, dogs with prior history of pregnancy can be used safely as blood donors. Conversely, no additional pretransfusion compatibility studies would be required should these dogs themselves need to be transfused.

Immune assessment of farm-reared Penaeus vannamei shrimp naturally infected by IMNV in NE Brazil

Farm-reared shrimp production was growing intensely in Brazil until 2002, when an outbreak of a new disease, caused by the infectious myonecrosis virus (IMNV), disrupted the production in the northeast region. The aim of this study was to evaluate some hemato–immunological parameters in Penaeus vannamei shrimp during the progress of the disease caused by IMNV. Affected shrimp were separated into three groups, according to disease severity: asymptomatic shrimp (Group AS), shrimp with initial symptoms (Group IN) and with advanced symptoms (Group AD). The parameters examined were: hemograms, percentage of apoptotic hemocytes (Hoechst staining), anion superoxide production (NBT reduction); phenoloxidase (PO) activity (L-DOPA oxidation), agglutinating titre (dog erythrocytes) and antimicrobial activity of the hemolymph. Significant changes in immunological parameters were only seen in Group AD shrimp, where increases in apoptotic hemocytes (8-fold), agglutinating titre (16 fold), anion superoxide hemocyte production (50%) and hemolymph antimicrobial activity (21-fold against Micrococcus luteus) were observed when compared to shrimp of Groups AS and IN. The group AD shrimp also showed significant reductions in total hemocyte counts (30%), percentage of circulating granulocytes (7%) and PO activity (50%). These results suggest that the immune system of IMNV-infected shrimp responds only at a late stage of the disease, when animal recovery is unlikely. This is the first study to examine the immune response of P. vannamei during the progress of disease caused by IMNV in Brazilian farms where the virus first appeared.

Cytotoxicity and hemolytic activity of jellyfish Nemopilema nomurai (Scyphozoa: Rhizostomeae) venom

The recent bloom of a giant jellyfish Nemopilema nomurai has caused a danger to sea bathers and fishery damages in the waters of China, Korea, and Japan. The present study investigated the cytotoxic and hemolytic activities of crude venom extract of N. nomurai using a number of in vitro assays. The jellyfish venom showed a much higher cytotoxic activity in H9C2 heart myoblast than in C2C12 skeletal myoblast (LC 50 = 2 µg/mL vs. 12 µg/mL, respectively), suggesting its possible in vivo selective toxicity on cardiac tissue. This result is consistent with our previous finding that cardiovascular function is a target of the venom. In order to determine the stability of N. nomurai venom, its cytotoxicity was examined under the various temperature and pH conditions. The activity was relatively well retained at low environmental temperature (≤ 20 °C) and dramatically lost at high temperature (≥ 60 °C). In pH stability test, the venom has abruptly lost its activity at low pH environment (pH ≤ 4). Interestingly enough, however, its activity was not significantly affected even at the highest pH environment tested (pH ≤ 12) in the present study. Additionally, hemolytic activity of the venom was examined using the erythrocytes of cat, dog, human, rabbit and rat. Venom concentration-dependent hemolysis could be observed from 10 µg/mL of protein equivalents or higher with variable potencies in different species, among which dog erythrocyte was the most susceptible to the venom (EC 50 = 151 µg/mL). SDS-PAGE analysis of N. nomurai venom showed the molecules of 20–40 kDa and 10–15 kDa appeared to be the major protein components of the venom.

Kinetic properties of dehydroascorbic acid transport in erythrocytes of young and adult beagle dogs

Kinetic properties of dehydroascorbic acid (DHA) transport were studied in erythrocytes of beagle dogs at 1 and 12 months. DHA uptake was much faster in erythrocytes of dogs at 1 than 12 months. Young dogs showed much greater affinity for DHA than adult dogs and the rate of DHA uptake was not affected by glucose. It was due to the presence of fetal erythrocytes with high activity of Glut-1 glucose transporter in young dogs. In adult dogs, DHA uptake was slow and competitively inhibited by physiological concentrations of glucose. Glut-4 was identified as the carrier protein responsible for DHA uptake in erythrocytes of adult dogs. Glut-4 was hardly detectable in neonatal dogs, but increased to adult levels by 3 months as fetal erythrocytes were eliminated and switched to adult cells.

Canine elliptocytosis due to a mutant β‐spectrin

A 5-year-old, spayed female, mixed-breed dog with persistent elliptocytosis was evaluated at the Veterinary Medical Teaching Hospital at Kansas State University. The elliptocytosis was asymptomatic and was detected during the evaluation of lameness. When subjected to shear stress in an ektacytometer, the dog's erythrocytes had reduced cellular deformability and erythrocyte membranes had decreased mechanical stability. Analysis of erythrocyte membrane spectrin by nondenaturing gel electrophoresis revealed an increased amount of spectrin dimers, indicating a defect in spectrin self-association. DNA analysis detected a beta-spectrin mutation in codon 2110 in which threonine was replaced by methionine. This mutation likely altered the molecular structure of the erythrocyte membrane, leading to impaired spectrin self-association and elliptocyte formation.

Age-dependent changes in uptake and recycling of ascorbic acid in erythrocytes of Beagle dogs

Uptake and recycling of ascorbic acid (AA) were studied in erythrocytes of 1- to 12-month-old Beagle dogs. At 1 month, both AA uptake and recycling capacity were high. Ascorbic acid entered erythrocytes mainly in the oxidized form with elevated activity of Glut 1 glucose transporter. However, this trait of erythrocytes was rapidly lost in the course of postnatal growth. At 3 months, ascorbic acid uptake and recycling capacity decreased to almost adult levels. Thereafter, AA was transported mainly in the reduced form, and its uptake and recycling capacity became one-third the levels of 1-month-old dogs. Postnatal anemia and recovery were indicated by changes in hemoglobin and packed cell volume levels at 1 and 3 months. Glutathione reductase (GR) activity was twice as high as in adults in 1-month-old dogs, allowing efficient reduction of oxidized ascorbic acid, which enters cells in large amounts due to elevated activity of the Glut 1 glucose transporter. One-month-old dogs need high levels of AA for antioxidant protection and skeletal development. The high AA recycling capacity of erythrocytes is considered to balance the expenditure of AA in young Beagle dogs.