Porcine Erythrocyte Membranes Research Articles

Biomembranes from slaughterhouse blood erythrocytes as prolonged release systems for dexamethasone sodium phosphate.

The present study investigated preparation of bovine and porcine erythrocyte membranes from slaughterhouse blood as bio-derived materials for delivery of dexamethasone-sodium phosphate (DexP). The obtained biomembranes, i.e., ghosts were characterized in vitro in terms of morphological properties, loading parameters, and release behavior. For the last two, an UHPLC/-HESI-MS/MS based analytical procedure for absolute drug identification and quantification was developed. The results revealed that loading of DexP into both type of ghosts was directly proportional to the increase of drug concentration in the incubation medium, while incubation at 37°C had statistically significant effect on loaded amount of DexP (P < 0.05). The encapsulation efficiency was about fivefold higher in porcine compared to bovine ghosts. Insight into ghosts' surface morphology by field emission-scanning electron microscopy and atomic force microscopy confirmed that besides inevitable effects of osmosis, DexP inclusion itself had no observable additional effect on the morphology of the ghosts carriers. DexP release profiles were dependent on erythrocyte ghost type and amount of residual hemoglobin. However, sustained DexP release was achieved and shown over 3 days from porcine ghosts and 5 days from bovine erythrocyte ghosts. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1046-1055, 2016.

The immune adherence receptor CR1-like existed on porcine erythrocytes membrane.

In the present study, we obtain a mouse anti-porcine complement receptor type 1 (CR1)-like monoclonal antibody (McAb) and use this McAb to verify the existence of CR1-like protein on porcine erythrocytes. Our results confirm that CR1-like protein is localized on the surface of porcine erythrocytes. Mouse immunoglobulin G inhibited the binding of serum-opsonized green fluorescent protein-expressing Escherichia coli to porcine erythrocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicates that CR1-like McAb reacts with biochemically-purified porcine erythrocyte membrane fractions, with a clear band at 135 kDa to 140 kDa. We postulate that the 135 kDa to 140 kDa membrane protein is the equivalent of the porcine erythrocyte CR1-like protein.

Identification of Mycoplasma suis MSG1 interaction proteins on porcine erythrocytes.

Adhesion protein MSG1 mediating adherence to porcine erythrocytes in Mycoplasma suis (M. suis) invasion has been identified previously. In order to determine the host membrane proteins that interact with MSG1, recombinant His-tagged MSG1 (rMSG1) was used to screen for interacting proteins in the protein extracts of porcine erythrocyte membrane. Potential rMSG1-interacting proteins were initially identified as band 3 and β-actin with molecular weight of 46 and 45 kDa, respectively. Immune fluorescence results showed that rMSG1 can specifically bind with the β-actin of HeLa, BHK-21, and HEK-293A cells, respectively. RNA interference assays further demonstrated that the interaction between β-actin and rMSG1 on HeLa cells was specific and dose dependent. Confocal microscopy showed that both rMSG1 and M. suis can partially co-localize with β-actin on the surface of porcine erythrocytes. Pull-down assays showed that rMSG1 can directly interact with β-actin. Our study is the first to report the interaction of MSG1 with β-actin, which will be of help to understand the pathogenesis of M. suis and develop a cultivation system.

BINDING OF HUMAN NATURAL ANTIBODIES TO NON??Gal XENOANTIGENS ON PORCINE ERYTHROCYTES1

Xenotransplantation is considered one of possible solutions for the serious shortage of organs and cells in transplantation. Although the alphaGal epitope (Gal alpha1,3Gal beta1,4GlcNAc-R) has been identified as being a major xenoantigen responsible for hyperacute rejection, the removal of anti-alphaGal antibody alone from human serum is insufficient to circumvent antibody-mediated immune responses. We report here the characterization of xenoreactive human natural antibodies against antigens without the alphaGal epitope (nonalphaGal xenoantigens) on porcine erythrocytes using flow cytometry and the evidence for their involvement in complement-mediated hemolysis. Furthermore, a novel protein of 45-kDa has been isolated from the porcine erythrocyte membrane as a major protein antigen recognized by human anti-nonalphaGal. The data presented here will add to our knowledge of xenoantigens on porcine red cells and be important for developing strategies to produce modified red cells immunologically compatible to humans.

Introduction to porcine red blood cells: Implications for xenotransfusion

Advances in the field of xenotransplantation raise the intriguing possibility of using porcine red blood cells ( pRBCs) as an alternative source for blood transfusion. The domestic pig is considered the most likely donor species for xenotransplantation. However, identification of xenoantigens on porcine erythrocytes and elucidation of their possible roles in antibody-mediated RBC destruction are necessary for developing clinical strategies to circumvent immunological incompatibility between humans and pigs. Although the αGal epitope (Galα1,3Galβ1,4GlcNAc-R) is the major xenoantigen on porcine erythrocytes and is responsible for the binding of the majority of human natural antibodies, other non-αGal xenoantigens have been identified. The importance of these non-αGal xenoantigens in binding human natural antibodies and subsequently triggering immunological responses cannot be underestimated. Our data suggest that non-αGal xenoantigen(s) identified on the porcine erythrocyte membrane are not only recognized by xenoreactive human natural antibodies but are also involved in complement-mediated hemolysis.

In vitro enzyme activation with calbindin-D 28k, the vitamin D-dependent 28 kDa calcium binding protein

Purified porcine erythrocyte membrane Ca 2+-ATPase and 3′:5′-cyclic nucleotide phosphodiesterase were stimulated in a dose-dependent, saturable manner with the vitamin D-dependent calcium binding protein from rat kidney, calbindin-D 28k (CaBP-D 28k). The concentration of CaBP-D 28k required for half-maximal activation ( K 0.5 act.) of the Ca 2+-ATPase was 28 nM compared to 2.2 nM for calmodulin (CaM), with maximal activation equivalent upon addition or either excess CaM or CaBP-D 28k, 3′:5′-Cyclic nucleotide phosphodiesterase (PDE) also showed equivalent maximum saturable activation by calbindin ( K 0.5 act. = 90 nM) or calmodulin ( K 0.5 act. = 1.2 nM). CaBP-D 28k was shown to effectively compete with CaM-Sepharose for PDE binding. Immunoprecipitation with CaBP-D 28k antiserum completely inhibited calbindin-mediated activation of PDE but had no effect on calmodulin's ability to activate PDE. While the physiological significance of these results remains to be established, they do suggest that CaBP-D 28k can activate enzymes and may be a regulator of yet to be identified target enzymes in certain tissues.

Human blood group glycosphingolipids of porcine erythrocytes

Two glycosphingolipids with human blood group A and H antigenicity were isolated from porcine erythrocyte membranes which were obtained from the pooled blood. The yield of the A- and H-antigenic glycolipids was approximately 0.2 and 0.1 % of total neutral glycolipids, respectively. No B antigen was detected. Through several methods the porcine erythrocyte antigens were all found to belong to lactoseries (type 1 chain), IV 2Fucα,IV 3GalNAcαLc 4Cer for type A and IV 2 FucαLc 4Cer for type H, in contrast to the antigenic glycolipids in human erythrocytes, which mostly belong to neolactoseries (type 2 chain). The constituent fatty acids of the A antigen were 75% normal acids and 25% 2-hydroxy acids, and the long chain base was 95% sphingenine. This is the first demonstration of the A-and H-antigenic glycolipids on erythrocytes of pig in whose gastric mucin the human blood group A and H substances have been demonstrated.

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.

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.

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.

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.

Effect of ionizing radiation on the transport of spin-labelled compounds across the porcine erythrocyte membrane. II.

Effect of ionizing radiation on the transport of spin-labelled compounds across the porcine erythrocyte membrane. II.

Comparison of the photodynamic action of Rose Bengal and tetrachlorosalicylanilide on isolated porcine erythrocyte membranes

The light-mediated effects of Rose Bengal and 3,3′,4′,5-tetrachlorosalicylanilide (T 4CS) on porcine erythrocyte membranes have been investigated. Irradiation in the presence of Rose Bengal and oxygen causes extensive destruction of the unsaturated fatty acids of the erythrocyte membrane. This results in a decrease in the membrane flexibility as measured by a nitroxide spin probe. Irradiation in the presence of T 4CS and oxygen had no measurable effect on the unsaturated fatty acids or on the membrane flexibility. Irradiation of erythrocyte membranes both in the presence of Rose Bengal and oxygen and of T 4CS gave rise to polymerisation of the membrane proteins. This was evident by polyacrylamide gel electrophoresis and by freeze-fracture electron microscopy. Aggregation of membrane proteins could be observed with low levels of Rose Bengal and short irradiation times at which no loss of unsaturated fatty acids could be detected. Irradiation of the n-butanol-extracted apoprotein of porcine erythrocyte membranes both in the presence of Rose Bengal and of T 4CS resulted in polymerisation of the protein as measured by gel electrophoresis and electron microscopy. The results obtained from the two photodynamic compounds are compared in terms of their different mechanisms of action on the membrane. The implications of the results in determining the molecular events leading to photohaemolysis are discussed.

Isolation and characterization of alkali-labile oligosaccharide units from porcine erythrocyte glycophorin.

The oligosaccharide units of glycophorin isolated from porcine erythrocyte membranes were released by alkaline borohydride treatment and purified by gel filtration and ion-exchange chromatography. Structures of the O-glycosidic oligosaccharides were determined by methylation analysis, the methylated sugar being identified by gas-liquid chromatography-mass spectrometry and nitrous acid deamination after hydrazinolysis. The major oligosaccharide was a trisaccharide, Gal(1 leads to 3)[Neu-NGly(2 leads to 6)]GalNAc. The other oligosaccharides were larger and contained Glc-NAc. One was a pentasaccharide, Gal(1 leads to 3)Gal(1 leads to 4)GlcNAc(1 leads to 3)Gal(1 leads to 3)GalNAc. The structure of the trisaccharide was also analyzed by direct-probe mass spectrometry of the permethylated derivative, and the result obtained was consistent with the proposed structure.

Comparison of isolated peanut agglutinin receptor glycoproteins from human, bovine and porcine erythrocyte membranes

Affinity chromatography has been used to isolate and compare the peanut agglutinin receptors from neuraminidase-treated human, bovine and porcine erythrocyte membranes. Passage of Triton X-100-solubilised membrane material through either Sepharose- or acrylamide-based affinity columns resulted in the reversible binding of receptor molecules to the immobilised lectin. Elution with 0.2M galactose released specifically bound glycoprotein fractions, the composition and molecular weights of which were determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Carbohydrate analysis by gas chromatography identified these bound glycoprotein fractions as the major sources of the O- glycosidic-linked disaccharide galactosyl-β-(1 → 3)-N- acetylgalactosamine in these membranes. It is suggested that these isolated fractions represent a discrete population of glycoproteins within the membranes studied, which possess both O- glycosidic - and N- glycosidic-linked carbohydrates.

Partial amino acid sequence of glycophorin from porcine erythrocyte membranes

Glycophorin from porcine erythrocyte membranes was digested with trypsin and chymotrypsin. Some of the peptides were isolated by conventional techniques. The amino acid sequence was determined for two isolated peptides: a chymotryptic glycopeptide of 19 residues and a tryptic peptide of 36 residues which represented the carboxy terminal of the glycophorin.

Some common characteristics of the acetylcholinesterase of human, bovine and porcine erythrocytes.

More than 80% of the acetylcholinesterase present in porcine erythrocyte membrane could be released from it by repeated washing with hypotonic buffer, as was true in the case of bovine erythrocyte. Density-gradient centrifugation revealed that the released enzyme particles of either species of mammals have a similar density of about 1.07 which is significantly lower than that of the original stroma. It has phospholipid and cholesterol contents higher than those of the stroma. Introduction of dinitrophenyl residue to the outer surface of the erythrocyte membrane caused about 60% inhibition of the acetylcholinesterase activity in all of human, bovine and porcine erythrocytes. Penicillin bound to the erythrocyte membrane exerted almost complete inhibition of the enzyme activity in all cases. These results suggest that acetylcholinesterase of similar nature is present in a form of lipoprotein on the outer surface of the erythrocyte membrane, with a density lower than that of the main part of the membrane.