Porcine Erythrocytes Research Articles

Characterization of neutral glycosphingolipids from porcine erythrocyte membranes

1. 1. Six neutral GSL fractions were purified from porcine erythrocyte membranes. 2. 2. They were identified to be LacCer (14% of total neutral GSLs), 2-hydroxy acid-rich and -poor Gb 3Cer (3 and 7%, respectively) and Gb 4Cer (71%) by means of NMR spectrometry. 3. 3. Monohexosylceramides (5%) were composed of GlcCer and GalCer with near amount. 4. 4. All these GSL classes contained a high concentration (more than 20% of total acids in each class) of 2-hydroxy fatty acids. 5. 5. GalCer and GlcCer contained considerable amounts of C16- and C18-acids, and of C18-phytosphingosine, whereas C24-acids and C18-sphingosine were predominant in the other GSLs. 6. 6. A minor GSL fraction (less than 1% of total neutral GSLs) which migrated more slowly than Gb 5Cer on a thin layer plate and composed of several GSL components contained L-fucose.

Isoenzyme patterns of glutathione transferases from mammalian erythrocytes.

The occurrence of glutathione transferase isoenzymes in mammalian erythrocytes was investigated. The enzymes present in the hemolysates of human, horse, beef, pig, and sheep erythrocytes were purified by a column of GSH-linked epoxy-activated Sepharose 6B and subjected to an isoelectric focusing run in the pH range 3.5-10. Human and horse preparations were resolved in a single peak of activity centered at pH 4.6 and 5.9, respectively. Two forms with a maximum of activity at pH 4.9 and 7.0 and four with a maximum at pH 5.9, 6.5, 7.1, and 8.1 were separated from bovine and porcine erythrocytes. At least six forms ranging from pH 4.3 to pH 7.1 were present in the ovine preparation, the neutral contributing more than 90% of total activity. The subunit composition of affinity-bound fractions was studied by sodium dodecyl sulfate-gel electrophoresis. The analysis revealed that erythrocyte glutathione transferases are composed of subunits of identical molecular weights. This result suggests that the polymorphism existing in beef, pig, and sheep may be due to charge isomers. The erythrocyte glutathione transferases did not express selenium-independent GSH peroxidase activity.

An improved hemagglutination test for study of canine parvovirus

Optimal conditions for hemagglutination (HA) by canine parvovirus (CPV) strains were investigated using several buffers. Porcine erythrocytes often agglutinated spontaneously in phosphate-buffered salt solution, isotonic saline solution or barbitone-complement-fixation buffer. Results were reproducible when borate-buffered saline (BBS) was used as the diluent for antigen, and “virus adjusting diluent” (VAD), containing 0.15 M NaCl and 0.3 M phosphate was used as the diluent for erythrocytes. Highest HA titers were obtained at pH 6.0 using BBS and VAD. Specific HA with CPV was observed not only at 4°C but at 37°C, and erythrocytes from horse, shrew mouse, hamster, cat, sheep and dog, as well as pig and African green monkey were agglutinated by CPV using the improved method.

Exposure of major neutral glycolipids in red cells to galactose oxidase. Effect of neuraminidase.

The exposure of several major red-cell glycolipids to galactose oxidase was studied by oxidizing the cells with the enzyme and reducing them with NaB2H4. After isolation, the deuterium label was detected by mass fragmentography. 60-70% globoside in human and porcine erythrocytes was exposed as measured by this method. In contrast, asialo-GM2 in guinea-pig erythrocytes and Forssman glycolipid in sheep erythrocytes were mainly in a cryptic state. Neuraminidase treatment increased the incorporation of deuterium label to asialo-GM2 4-8-fold. A similar effect was seen in Forssman glycolipid when sheep red cells were labeled with the neuraminidase/galactose oxidase/NaB3H4 method. In contrast, the increase in labeling was only about 10-40% in porcine and human globosides, which were efficiently exposed to galactose oxidase already in native red cells.

Effect of thiol reactive reagents and ionizing radiation on the permeability of erythrocyte membrane for non-electrolyte spin labels.

The paper presents some results on the effect of PCMB and NEM on the transport of non-electrolyte spin labels: TEMPO and TEMPOL across non-irradiated and irradiated porcine erythrocyte. Irradiated erythrocytes exhibited increased inhibitory effect of thiol reactive compounds in the TEMPO and TEMPOL transport compared to non-irradiated erythrocytes.

Prevention by antioxidants of the hemolysis of erythrocytes of cattle, pigs and humans treated with t-butyl hydroperoxide

1. Urate, 3-ribosylurate, ascorbate, glutathione and plasma protected bovine, porcine and human erythrocytes from hemolysis caused by t-butyl hydroperoxide (t-BHP). 2. Urate partially protected porcine erythrocytes from hemolysis by t-BHP when it was added 15 min after the addition of the t-BHP, but it did not protect when added 30 min after the t-BHP. 3. Glutathione and ascorbate protected oxyhemoglobin from oxidation to methemoglobin by t-BHP; 3-ribosylurate gave only slight protection. 4. Urate stimulated the formation of methemoglobin from oxyhemoglobin during treatment with t-BHP.

Characterization of gangliosides of porcine erythrocyte membranes: occurrence of ganglioside GD3 as major ganglioside.

Four major ganglioside species were isolated from porcine erythrocyte membranes by DEAE-Sephadex and Iatrobeads column chromatography. Treatment of the lipids with graded neuraminidase and beta-galactosidase, gas chromatographic analysis of their carbohydrates, sphingosine bases and molecular species of sialic acid revealed that the structure of these gangliosides were GM3(NeuAc), GM3(NeuGc), GD3(NeuAc) and GD3(NeuGc), each of which was 16 +/- 2 micrograms, 304 +/- 42 micrograms, 30 +/- 3 micrograms and 240 +/- 26 micrograms, respectively, per gram of the dry erythrocyte stroma. The amount of GM3 and GD3 accounted for more than 95% of total gangliosides of the erythrocytes. Porcine erythrocytes may provide a good source for large scale preparation of ganglioside GD3 which recently was identified as a human melanoma-associated antigen.

Primary structure of porcine Cu,Zn Superoxide dismutase

The complete amino acid sequence of Cu,Zn Superoxide dismutase from porcine erythrocytes has been determined. Comparison of the sequence with that of the bovine enzyme shows an overall high degree of homology with conservation of the crucial residues and the presence of two regions prone to variation. In one of these hypervariable regions the insertion of one residue with respect to the bovine enzyme and evidence of structural microheterogeneity has been observed. On the basis of the three-dimensional structure of the bovine enzyme no obvious relationship is apparent between a specific amino acid replacement and the unique pH-dependence pattern of the activity of the porcine enzyme.

Comparison of the membrane-bound (Ca 2+ + Mg 2+)-ATPase in erythrocyte ghosts from some mammalian species

1. 1. The properties of the membrane-bound calcium-pumping protein, the (Ca 2+ + Mg 2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) were compared in erythrocyte ghosts isolated from five mammalian species—human ( Homo sapiens), bovine ( Bos taurus), porcine ( Sus scrofa malitensis), ovine ( Ovis aries crassicandus) and caprine ( Capra hircus syriaca). 2. 2. The specific activity of the enzyme in porcine erythrocytes is one order of magnitude higher than in the other species. It was also stimulated to various extents by the regulator protein, calmodulin, and by phosphatidylinositol in all the species. 3. 3. Analysis of membrane proteins revealed a number of differences which seem to suggest that the molecular architecture of the red cell membrane influences the activity of the enzyme.

59] Glutathione reductase

Publisher Summary Glutathione reductase is a flavoprotein catalyzing the NADPH-dependent reduction of glutathione disulfide (GSSG) to glutathione (GSH). The reaction is essential for the maintenance of glutathione levels. Glutathione has a major role as a reductant in oxidation–reduction processes, and serves in detoxication and several other cellular functions of great importance. A purification method of this enzyme from calf liver and rat liver is described in this chapter. Similar methods are used for the purification of the enzyme from yeast, porcine, and human erythrocytes. All the steps are carried out at about 5°. The purification method from calf liver consists of various steps including preparation of cytosol fraction, chromatography on DEAE-sephadex, precipitation with ammonium sulfate, and chromatography on hydroxyapatite. The purification of glutathione reductase from rat liver is usually combined with the preparation of glutathione transferases, thioltransferase, and glyoxalase I.

Effects of derivatives of hydroxypyruvaldehyde phenylosazone on the Ca2+-adenosine triphosphatase activity of porcine erythrocyte membrane.

Thirteen derivatives of hydroxypyruvaldehyde phenylosazone were tested for their effects on the Ca2+-adenosine triphosphatase (ATPase) activity of porcine erythrocyte membranes. Seven drugs including hydroxypyruvaldehyde phenylosazone itself and the m-CH3, p-CH3, o-Cl, p-Cl, 3, 4-di-Cl and p-Br derivatives, inhibited Ca2+-ATPase activity, while other derivatives such as o-CH3, 2, 4-di-Cl, 2, 5-di-Cl, o-NO2, m-NO2 and p-NO2 derivatives were essentially without effect. Hydroxypyruvaldehyde p-chlorophenylosazone was the most potent inhibitor, inhibiting the enzyme activity completely at 1 mM, whereas carbonylcyanide m-chlorophenylhydrazone, a typical inhibitor of Ca2+-ATPase, inhibited the activity by 85% at the same concentration.

Thermochemiluminescent assay of porcine, rat, and human erythrocytes for antioxidative deficiencies

The thermal induction of chemiluminescence of luminol-horseradish peroxidase-labeled erythrocytes from pigs, rats, and man was studied. The luminescent responses of rat, porcine, and human erythrocytes to heating were linear in respect to logs of counts per minute versus temperature. Landrace-Duroc crossbred pigs with a history of malignant hyperthermia (porcine stress syndrome) and Poland-China-miniature pigs inbred for malignant hyperthermia (MH) yielded erythrocytes with high-level thermochemiluminescence (TCL). Sprague-Dawley rat erythrocytes were intermediate in their production of TCL. Normal human and MH-resistant miniature swine erythrocytes produced low-level TCL. However, pretreatment of human erythrocytes with 1-chloro-2,4-dinitrobenzene (CDNB) resulted in high-level TCL. Furthermore, halothane enhanced the TCL of CDNB-treated human erythrocytes and Landrace-Duroc porcine erythrocytes that were not treated with CDNB. Red blood cells from pigs susceptible to the porcine stress syndrome demonstrated a TCL response very similar to CDNB-treated erythrocytes.

Haemagglutination ofBacteroides fragilis

The relationship between the ability to cause haemagglutination (HA) and the presence of capsules and/or pili was examined for 50 clinical isolates of Bacteroides fragilis. HA was tested using a slide technique, and bovine, porcine, guinea pig, rat, rabbit, horse, human, chicken and pigeon erythrocytes. Chicken and pigeon erythrocytes were the best indicators for HA with 43 (86%) of the strains tested causing HA and 39 (78%) with strong reactions. Capsule staining showed that the same 43 strains causing HA also produced a demonstrable capsule. No pili were found on either encapsulated or non-encapsulated strains using transmission electron microscopy. These results suggest that adherence of B. fragilis is related to the presence of capsular material, not pili.

Reconstitution of calpain I and calpain II from their subunits: Interchangeability of the light subunits

Calpain (Ca 2+-dependent cysteine proteinase) is known to be a heterodimer, composed of one heavy (called 80K) and one light (called 30K) subunit. Calpain I, a low-Ca 2+-requiring form from porcine and human erythrocytes, and calpain II, a high-Ca 2+-requiring form from porcine kidney, were separated into their respective 80K and 30K subunits by high-performance liquid chromatography on a TSK-Gel G3000SW column in 1 m NaSCN. The isolated 80K subunits from porcine calpains I and II showed Ca 2+-dependent proteolysis, though much depressed and requiring higher Ca 2+ concentrations compared with the respective parent calpains. The optimal conditions were set for the reconstitution of a heterodimeric molecule from the once denatured and separated 80K and 30K subunits. In such reconstitution, the 30K subunits of calpains I and II were found to be functionally identical and interchangeable; either a homologous or heterologous 30K subunit lowered the Ca 2+ requirement of an 80K subunit significantly and enhanced the proteolytic activity. Subunit interchange could also be demonstrated between porcine and human calpains.

Hemagglutinating factor in an extract of sarcoptes scabiei var. suis (de geer)

A naturally occurring hemagglutinating factor to tanned human O positive, ovine and porcine erythrocytes was found in extracts from Sarcoptes scabiei var. suis. This hemagglutinating factor did not react with bovine, equine or avian erythrocytes. This factor was demonstrated by microscopic examination of the tanned erythrocytes and by the passive hemagglutination assay.

Properties of acyl-coenzyme A:1-acylglycerophosphate acyltransferase and lipases in porcine erythrocyte membranes.

Acyl-CoA:1-acylglycerophosphate acyltransferase activity was found in porcine erythrocyte membranes. However, the membrane preparations did not catalyze the acylation of either 2-acylglycerophosphate or 2-acylglycerophosphocholine. The 1-acylglycerophosphate acyltransferase and the known acyl-CoA:1-acylglycerophosphocholine acyltransferase systems differ in their specificities for acyl-CoAs and in their stabilities to detergents. Diacylglycerol lipase and monoacylglycerol lipase activities were also detected in porcine erythrocytes. These two activities appear to be catalyzed by different enzymes inasmuch as diacylglycerol lipase but not monoacylglycerol lipase was completely inhibited by divalent cations. The diacylglycerol lipase was relatively specific for the 1-position yielding 2-acylglycerol. The monoacylglycerol lipase hydrolyzed 1-acylglycerol and 2-acylglycerol at comparable rates. Phosphatidic acid was dephosphorylated to form 1,2-diacylglycerol but the acyl groups of phosphatidate were not hydrolyzed significantly by the erythrocyte membranes. Thus, the origin of 1-acylglycerophosphate, a substrate for the newly described enzyme, acyl-CoA:1-acylglycerophosphate acyltransferase, in mature erythrocyte could not be ascribed to action of diacylglycerol lipase, glycerophosphate acyltransferase, or phosphatidate-specific phospholipase A. 1-Acylglycerophosphate may be supplied extracellularly or the 1-acylglycerophosphate acyltransferase activity in erythrocytes may be a remnant of de novo phosphatidate synthesizing system of reticulocytes.

K99 surface haemagglutinin of enterotoxigenic E. coli recognize terminal n-acetylgalactosamine and sialic acid residues of glycophorin and other complex glycoconjugates

The possible nature of the erythrocyte receptor for K99 haemagglutinin is described. The receptor is a glycophorin which possesses NANA α 2–6 GalNAc structures for which this haemagglutinin has a strong affinity. Human, porcine and equine erythrocytes which are haemagglutinated by K99 all have similar glycophorin oligosaccharide structures. K99 has affinity for both NANA and GalNAc at terminal positions, but the affinity for α 2–6-linked NANA is higher than for α 2–8-linked NANA.

Detection of anti-zona pellucida activities in human sera by the passive hemagglutination reaction

A simple method of detecting anti-zona activities in human sera has been developed by passive hemagglutination reaction ( PHAR ) using bovine erythrocytes as indicator cells coated with purified porcine zona substance. By this test, 8 of 88 serum samples (9.1%) from infertile women gave positive reactions, whereas only 1 of 90 control sera (1.1%). A positive immunofluorescence (IF) assay confirmed the results of the PHAR in seven of the eight positive samples. Positive PHAR and IF on porcine zonae were abolished after adsorption with porcine erythrocytes, but IF on human zonae was retained by four of eight serum samples, indicating the presence of anti-zona autoantibodies in these infertile sera that were directed to an antigen(s) proper to human zonae.

Differences in C-terminal amino acid sequences between erythrocyte and liver cytochrome b5 isolated from pig and human : Evidence for two tissue-specific forms of cytochrome b5

Two forms of cytochrome b 5, a soluble erythrocyte form and a membrane-bound liver form, were purified from pig and human, and structural differences between them were analyzed. Porcine and human erythrocyte cytochrome b 5 consisted of 97 amino acid residues and contained the same catalytic domain structure (residue 1–96) as that of the corresponding liver cytochrome b 5, but had one amino acid replacement at the C-terminus (residue 97). These results suggest that erythrocyte cytochrome b 5 is not derived from the liver protein by proteolysis but a translational product from another distinct mRNA of cytochrome b 5.

In vitro studies on feline panleucopaenia virus. Standardisation of haemagglutination-inhibition test for feline panleucopaenia virus antibody

Standardised procedure for obtaining reproducible haemagglutination-inhibition results for FPV antibody which correlate with serum-neutralization titres was described. Optimal conditions were found to be Alsevers anticoagulant, PBS/0.05% BSA (pH 6.8) as buffer, especially washed round bottom microplates, determination of maximally sensitive porcine erythrocytes, use of reproducible erythrocyte concentrations, inactivation of serum samples at 56°C for 30 min and serum treatment with koalin pH 9.0. The concentration of erythrocyte used for estimation of haemagglutination units in H1 test should not differ from that used as indicator in the test. Predilution of serum beyond 1:4 associated with false results. Reproducible method for removing natural agglutininus in serum by adsorbtion with erythrocytes was described.