Isolation and partial characterization of two minor glycoproteins from human erythrocyte membranes

Two minor glycoproteins GP-II and GP-III, were isolated from human erythrocyte membranes and characterized chemically and immunologically. The chemical composition of GP-II and GP-III was similar: GP-II consisted of 81% protein and 19 % carbohydrate of which 4.9 % was hexose. 5.4 % hexosamine and 7.8 % sialic acid. GP-III consisted of 76 % protein and 24 % carbohydrate of which 7.6 % was hexose, 7.2 % hexosamine and 8.1 % sialic acid. The amino acid composition of GP-II and GP-III was also similar. GP-II and GP-III, however, differed in chemical composition from the MN glycoprotein. GP-II and GP-III were associated with the blood group activities Ss, I and A, but not with the MN antigens. GP-III had higher blood group activities per μg of protein than did GP-II. The specific activities for the Ss blood group antigens were increased 3–10-fold by purification of GP-III from the aqueous phase of chloroform methanol extracts.     

Characterization of the Ss sialoglycoprotein and its antigens in Rhnull erythrocytes

The Ss sialoglycoprotein (glycophorin B) and its antigens in Rhnull erythrocytes, which lack the Rhesus blood group antigens, due to apparently silent (amorphic type) or independent suppressor (regulator type) genes, were investigated. The quantity of the molecule in amorphic and in regulator type red cell membranes was found to be decreased by about 60%-70%, as judged from sodium-dodecylsulfate polyacrylamide gel electrophoresis. The Ss glycoprotein content in the erythrocytes from heterozygotes (regulator type) was diminished to an extent of about 30%. Confirming and extending previous studies, the S, s, Ux, Uz and 'N' antigens were slightly weakened in Rhnull erythrocytes. The U and Duclos receptors were only slightly or not depressed in amorphic Rhnull cells, but almost absent from or not detectable in those of the regulator type. This demonstrates that an additional alteration, apart from the decreased Ss glycoprotein content of the membranes, accounts for the weakness of these receptors in regulator type cells. We propose the hypothesis that (a) protein(s) encoded by the Rhesus locus form(s) a complex with the Ss glycoprotein. Thus, it (they) might facilitate the incorporation of the Ss glycoprotein into the membrane and also contribute to the complete expression of the U and Duclos antigens in normal cells.     

Structure of the Ss blood group antigens, II: a methionine/threonine polymorphism within the N-terminal sequence of the Ss glycoprotein

The N-terminal amino acid sequence (residues 1--35) of the Ss sialoglycoprotein (or glycophorin B) from human erythrocyte membranes of defined Ss blood group activity was determined by manual sequencing methods, using N-terminal tryptic or chymotryptic glycopeptides and various secondary peptides. The proposed structure differs considerably from that suggested on the basis of work with glucopeptides of unknown Ss blood group activity (Furthmayr, Nature 271, 519--523, 1978). Only one difference between glycopeptides from Ss and ss erythrocytes was found, i.e. a methionine/threonine polymorphism at position 29. On the basis of previous work (Dahr et al., Hoppe-Seyler's Z. Physiol. Chem. 361, 145--152, 1980), it is concluded that this amino acid heterogeneity represents the Ss polymorphism rather than the UX or UZ polymorphisms, which are in strong genetic linkage disequilibrium with the Ss antigens. A part of the sequence (residues 9--30) of the major (MN) red cell membrane sialoglycoprotein (glycophorin A) was re-investigated and revised at positions 11 and 17. As judged from the present data, the first 26 residues of the Ss and the blood group N-specific MN glycoprotein are identical. The sequence 27--35 of the Ss glycoprotein shows a homology with the residues 56--64 and 59--67 of the MN glycoprotein. Data on the partial N-terminal sequence of glycopeptides from a third erythrocyte membrane sialoglycoprotein (component D or glycophorin C) indicate that its structure is different from those of the two other glycoproteins.     

An RFLP for glycoprotein A (MN) is in linkage disequilibrium with MN and Ss

Using a cDNA for glycophorin A (MN), we screened 10 unrelated Caucasians using 22 restriction enzymes for RFLPs. A common StuI RFLP was identified and shown to be in marked linkage disequilibrium with both the MN and Ss blood-group antigens in a larger group of unrelated Caucasians. This provides a DNA marker for a locus that has been of major importance in genetic and population studies. The demonstrated disequilibrium will prove useful in localizing the gene for glycophorin B and in studies of genetic and physical distances on human chromosomes.     

Murine antigen H-2.7: localization of its antigenic determinant to the Ss (C4) molecule

Murine blood group antigen H-2.7 is encoded by a locus mapping in the vicinity of the S locus which codes for the Ss antigen carried by the fourth component of the complement pathway (C4). Normal mouse serum of H-2.7-positive strains contains a substance which inhibits anti-H-2.7 hemagglutination. This substance cannot be removed by passage of the serum through an anti-Ss immunoabsorbent column indicating that the Ss and H-2.7 antigens are present on separate molecules or molecular fragments in the serum. In contrast, fresh plasma either does not contain the H-2.7-bearing substance at all or it contains it at a far lower concentration than normal serum, although it has a normal level of the Ss-antigen-bearing substance. However, the H-2.7-positive substance appears when the plasma is allowed to stand for several hours, or when it is dialyzed and treated subsequently in a manner favoring spontaneous degradation of complement components. Removal of the Ss substance from the fresh plasma prevents the appearance of the H-2.7 antigen at any time thereafter. These findings indicate that the Ss and H-2.7 antigens are carried by the same molecule or molecular complex. The intact molecule expresses only the Ss antigen; the H-2.7 antigen is either hidden or masked so that it is inaccessible or poorly accessible to H-2.7 antibodies. Degradation of these molecules results in the generation of two fragments, a large fragment carrying the Ss antigen and a smaller H-2.7-positive fragment. The data are consistent with the interpretation that the H-2.7 antigen is encoded by the S locus, and that it is carried by that portion of the C4 molecule split off during complement activation.     

Quantitative variations in the expression of the mouse serum antigen Ss and its sex-limited allotype Slp

A radial immunodiffusion assay for quantitation of the Ss and Slp serum antigens is described. Significant differences between the mean serum concentrations of Ss and Slp were found among various inbred strains. Some of these differences have been shown to be associated with the H-2 haplotype. The quantitative difference between Slp levels associated with the H-2 ^a and H-2 ^S haplotypes has been used as a marker for the S region in the analysis of certain H-2 recombinant strains [A.TH, B10.S(7R), B10.S(9R), and B10.BSVS]. Male mice of two strains with the H-2 ^b haplotype have been shown to have significantly lower levels of Ss compared to males of the other strains tested. Male mice of every strain examined were found to have significantly higher levels of Ss in their serum than females of the same strain. The molecular relationship and developmental patterns of the Ss and Slp antigens have also been investigated using the radial immunodiffusion assay.     

Murine antigen H-2.7: Localization of its antigenic determinant to the Ss (C4) molecule

Murine blood group antigen H-2.7 is encoded by a locus mapping in the vicinity of theS locus which codes for the Ss antigen carried by the fourth component of the complement pathway (C4). Normal mouse serum of H-2.7-positive strains contains a substance which inhibits anti-H-2.7 hemagglutination. This substance cannot be removed by passage of the serum through an anti-Ss immunoabsorbent column indicating that the Ss and H-2.7 antigens are present on separate molecules or molecular fragments in the serum. In contrast, fresh plasma either does not contain the H-2.7-bearing substance at all or it contains it at a far lower concentration than normal serum, although it has a normal level of the Ss-antigen-bearing substance. However, the H-2.7-positive substance appears when the plasma is allowed to stand for several hours, or when it is dialyzed and treated subsequently in a manner favoring spontaneous degradation of complement components. Removal of the Ss substance from the fresh plasma prevents the appearance of the H-2.7 antigen at any time thereafter. These findings indicate that the Ss and H-2.7 antigens are carried by the same molecule or molecular complex. The intact molecule expresses only the Ss antigen; the H-2.7 antigen is either hidden or masked so that it is inaccessible or poorly accessible to H-2.7 antibodies. Degradation of these molecules results in the generation of two fragments, a large fragment carrying the Ss antigen and a smaller H-2.7-positive fragment. The data are consistent with the interpretation that the H-2.7 antigen is encoded by the S locus, and that it is carried by that portion of the C4 molecule split off during complement activation.Abbreviations used in this paper NMS normal mouse serum - DNP dinitrophenyl - EDTA ethylene-diamine-tetraacetate - PVP polyvinylpyrrolidone     

Structural properties of the human MN blood group antigen receptor sites.

It is shown that the MN blood group antigen determinant of the major human erythrocyte membrane (MN) sialoglycoprotein is located on its N-terminal octaglycopeptide. The only analytically detectable difference between peptides from MM and NN cells are Ser/Leu and Gly/Glu polymorphisms at the first and fifth positions, respectively. Destruction of the antigens by removal of the N-terminal residues suggests that these amino acids represent a part of the receptor areas for various anti-M or -N reagents. Evidence is presented that the N-terminal structure of the Ss glycoprotein is identical with that of MN glycoprotein from NN red cells up to the fifth residue. This provides an explanation for the 'N' antigen on this molecule and direct support for the earlier proposal that the MNSs locus is represented by homologous genes.     

Strongy Detect: Preliminary Validation of a Prototype Recombinant Ss-NIE/Ss-IR Based ELISA to Detect Strongyloides stercoralis Infection

BackgroundStrongyloides stercoralis (Ss) is the etiological agent of strongyloidiasis, a neglected tropical disease of global concern. Laboratory diagnosis of strongyloidiasis is most often based on detection of antibodies against antigens in an enzyme linked immunosorbent assay (ELISA). Herein, we report a preliminary validation study of newly developed IgG4- and/or IgG- based ELISAs to detect strongyloidiasis (Strongy Detect, InBios) incorporating a cocktail of 2 previously described recombinant antigens, Ss-NIE and Ss-IR.MethodsThe sensitivity and specificity were determined by using the assay in 150 cryopreserved serum samples from humans known to be Ss infected (n = 74), helminth uninfected (n = 47), or infected with a helminth other than Ss [n = 29). The treatment associated dynamics of antibody detection were then assessed using 35 paired samples obtained before and after definitive therapy.ResultsThe IgG and IgG4 assays were 99% and 96% sensitive, respectively, and 99% and 100% specific, respectively. Neither the IgG or IgG4 assay showed cross reactions with sera from those infected with other helminths. Although ELISA values did decline post-treatment few returned to levels below the cutoff for infection.ConclusionStrongy Detect is the most sensitive and specific commercialized immunoassay for detection of strongyloidiasis. The assay remains positive for greater than a year post-treatment.     

IL-10- and TGFβ-mediated Th9 Responses in a Human Helminth Infection

Background

Th9 cells are a subset of CD4⁺ T cells that express the protoypical cytokine, IL-9. Th9 cells are known to effect protective immunity in animal models of intestinal helminth infections. However, the role of Th9 cells in human intestinal helminth infections has never been examined.

Methodology

To examine the role of Th9 cells in Strongyloidis stercoralis (Ss), a common intestinal helminth infection, we compared the frequency of Th9 expressing IL-9 either singly (mono-functional) or co-expressing IL-4 or IL-10 (dual-functional) in Ss-infected individuals (INF) to frequencies in uninfected (UN) individuals.

Principal Findings

INF individuals exhibited a significant increase in the spontaneously expressed and/or antigen specific frequencies of both mono- and dual-functional Th9 cells as well as Th2 cells expressing IL-9 compared to UN. The differences in Th9 induction between INF and UN individuals was predominantly antigen-specific as the differences were no longer seen following control antigen or mitogen stimulation. In addition, the increased frequency of Th9 cells in response to parasite antigens was dependent on IL-10 and TGFx since neutralization of either of these cytokines resulted in diminished Th9 frequencies. Finally, following successful treatment of Ss infection, the frequencies of antigen-specific Th9 cells diminished in INF individuals, suggesting a role for the Th9 response in active Ss infection. Moreover, IL-9 levels in whole blood culture supernatants following Ss antigen stimulation were higher in INF compared to UN individuals.

Conclusion

Thus, Ss infection is characterized by an IL-10- and TGFβ dependent expansion of Th9 cells, an expansion found to reversible by anti-helmintic treatment.     

Effects of inter-trial interval length on food-hoarding partial reinforcement of running behaviour in the golden hamster

Food-hoarding provides an adequate motivation in sated hamsters for the acquisition of a two-way running response. This learning was studied using a discrete-trial procedure, in continuous (CR) and partial reinforcement (PR) conditions, with two different inter-trial interval (ITI) lengths. The dependent variables were: the time spent by Ss in the goal section of the runway, and the number of their runs during extinction.The PR training had two effects on goal times: a slow decrease in acquisition on non-reinforced trials, and then a stabilization at this level during the extinction phase, as contrasted with the sudden increase found in CR-trained Ss when they were switched from acquisition to extinction conditions. However, the PR effects on number of runs depended upon ITI length: resistance to extinction of PR-trained Ss was superior to that of CR-trained Ss with spaced, but not with massed, trials. In the latter case, CR-trained Ss persisted as much as did PR-trained Ss. An hypothesis is offered, along the lines of the Frustration theory.     

人红细胞膜血型糖蛋白的研究进展

血型糖蛋白(GP)是红细胞膜中主要含唾液酸的跨膜蛋白,有A、B、C和D四种.GPA是MN血型糖蛋白,GPB表达Ss、U血型,GPC、GPD则是Gerbich抗原,四种血型糖蛋白的结构有不同程度的同源性,尤以同类间的同源性程度最高,GPA在防止红细胞之间、红细胞与血管内皮细胞之间的相互作用有重要功能,并在配体诱导下影响红细胞膜的物理性质.GPC是维持红细胞正常形状、正常物理性质的重要因子.GPA和GPC的功能还分别与带3蛋白、带4.1蛋白有关.     

Mapping and characterization of the mouse and human SS18 genes, two human SS18-like genes and a mouse Ss18 pseudogene.

We have previously isolated and characterized a mouse cDNA orthologous to the human synovial sarcoma associated SS18 (formerly named SSXT and SYT) cDNA. Here, we report the characterization of the genomic structure of the mouse Ss18 gene. Through in silico methods with sequence information contained in the public databases, we did the same for the human SS18 gene and two human SS18 homologous genes, SS18L1 and SS18L2. In addition, we identified a mouse Ss18 processed pseudogene and mapped it to chromosome 1, band A2-3. The mouse Ss18 gene, which is subject to extensive alternative splicing, is made up of 11 exons, spread out over approximately 45 kb of genomic sequence. The human SS18 gene is also composed of 11 exons with similar intron-exon boundaries, spreading out over about 70 kb of genomic sequence. One alternatively spliced exon, which is not included in the published SS18 cDNA, corresponds to a stretch of sequence which we previously identified in the mouse Ss18 cDNA. The human SS18L1 gene, which is also made up of 11 exons with similar intron-exon boundaries, was mapped to chromosome 20 band q13.3. The smaller SS18L2 gene, which is composed of three exons with similar boundaries as the first three exons of the other three genes, was mapped to chromosome 3 band p21. Through sequence and mutation analyses this gene could be excluded as a candidate gene for 3p21-associated renal cell cancer. In addition, we created a detailed BAC map around the human SS18 gene, placing it unequivocally between the CA-repeat marker AFMc014wf9 and the dihydrofolate reductase pseudogene DHFRP1. The next gene in this map, located distal to SS18, was found to be the TBP associated factor TAFII-105 (TAF2C2). Further analogies between the mouse Ss18 gene, the human SS18 gene and its two homologous genes were found in the putative promoter fragments. All four promoters resemble the promoters of housekeeping genes in that they are TATA-less and embedded in canonical CpG islands, thus explaining the high and widespread expression of the SS18 genes.     

Detection of stage specific and organ specific human brain antigens during embryogenesis]

The rabbit antisera were obtained against the water soluble antigens of the brain of 8--10 weeks old human foetuses. Three groups of specific antigens were identified in the brain of human foetuses: 1) antigens common for the embryonic brain and other organs of the same age; 2) antigens common for the embryonic brain and some organs of the adult organism; 3) stage (phase)-specific brain antigens present only in the brain between the 8th and 10th weeks of pregnancy.     

Alteration of the genes for glycophorin A and B in glycophorin-A-deficient individuals

Glycophorins A and B are homologous glycoproteins of the red cell membrane which carry the blood-group MN and Ss antigens, respectively, and are encoded by two distinct genes closely linked on chromosome 4, which are probably derived from each other by duplication during evolution. The lack of glycophorin A is associated with the rare phenotype En(a-), indicating individuals who are defective for MN antigens, as well as for the Ena antigens, also located on this glycoprotein. The En(a-) condition is heterogenous and includes two categories of variants exemplified by the Finnish and the English types referred to as En(Fin) and En(UK), respectively. By Southern blot and preliminary genomic clone analyzes we have compared the status of the genes for glycophorins A and B, as well as that of the gene encoding glycophorin C, another unrelated red cell membrane glycoprotein, in the En(a-) variants and in the En(a+) control donors. Our data indicate that the En(Fin) variant is homozygous for a complete deletion of the glycophorin A gene without any detectable abnormality of the genes encoding glycophorins B or C. In the genome of the En(UK) variant, with the presumed genotype Mk/En(UK), and where the Mk condition abolishes the expression of MN and Ss antigens, we have identified several abnormalities of the glycophorin A and B genes, but the glycophorin C gene was unaffected. Our results strongly support the view that in Mk chromosome the glycophorin A and B genes are largely deleted, whereas the En(UK) chromosome probably contains a gene fusion product encoding a hybrid glycoprotein AM-B, composed of the N-terminal portion of a blood group M-type glycophorin A and of the C-terminal portion of glycophorin B. The determination of the 5' and 3' limits of the hybrid gene and elucidation of the mechanism involved will require sequencing of the rearranged DNA of the variant and a full knowledge of the organization of the glycophorin A and B genes.     

Identification of tumour antigens by serological analysis of cDNA expression cloning

The identification of antigens that distinguish normal cells from cancer cells is an important challenge in the field of tumour immunology and immunotherapy. The immunoscreening of cDNA expression libraries constructed from human tumour tissues with antibodies in sera from cancer patents (SEREX: serological identification of antigens by recombinant expression cloning) provides a powerful approach to identify immunogenic tumour antigens. To date, over 2,000 tumour antigens have been identified from a variety of malignancies using SEREX. These antigens can be classified into several categories, of which the cancer/testis (CT) antigens appear to be the most attractive candidates for vaccine development. The SEREX-defined tumour antigens facilitate the identification of epitopes (antigenic peptides) recognised by antigen-specific cytotoxic T lymphocytes (CTLs) and provide a basis for peptide vaccine and gene therapy in a wide variety of human cancers. Moreover, some of these antigens seem to play a functional role in the pathogenesis of cancer.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Heart rate response as indicator of mental strain: interaction of personality and situational factors

Relation between mental strain defined as heart rate deviation score from person's baseline and persistence-excitation concept based on the Eysenck personality theory under various density information flow was investigated. Two groups the HPE(high EPQ-N and low EPQ-E score) and the LPE(low EPQ-N and high EPQ-E score) of a nuclear power plant operators were investigated under a monotonous condition (HPE: 15 Ss; LPE:12 Ss) and under a high density information flow condition (HPE: 17Ss, LPE: 15 Ss). The data support the view that the high persistence-low inhibition individual will be more mentally strained under high density information flow and that the level of strain will decelerate more slowly in comparison to persons with low persistence-high inhibition EPQ characteristics. The low persistence-high inhibition individuals will experience higher mental strain under monotonous situations. The findings suggest that the average EPQ-E and EPQ-N score might be useful criterion for selecting persons for tasks with alternatively monotonous and cognitively demanding situations.     

Immunohistochemical Demonstration of Specific Antigens in the Human Brain Fixed in Zinc-ethanol-Formaldehyde

Tissue fixation is critical for immunohistochemistry. Recently, we developed a zinc-ethanol-formalin fixative (ZEF), and the present study was aimed to assess the applicability of the ZEF for the human brain histology and immunohistochemistry and to evaluate the detectability of different antigens in the human brain fixed with ZEF. In total, 11 antigens were tested, including NeuN, neuron-specific enolase, GFAP, Iba-1, calbindin, calretinin, choline acetyltransferase, glutamic acid decarboxylase (GAD65), tyrosine hydroxylase, synaptophysin, and α-tubulin. The obtained data show that: i) the ZEF has potential for use in general histological practice, where detailed characterization of human brain morphology is needed; ii) the antigens tested are well-preserved in the human brain specimens fixed in the ZEF.Key words: Fixation, zinc-ethanol-formalin, immunohistochemistry, human brain     

Studies on Mv red cells. II. Immunochemical investigations

The properties of the Mv antigen, a low incidence receptor of the MNSs blood group system, were investigated by serological tests with protease treated red cells and inhibition assays with glycoproteins or peptides from normal and Mv erythrocytes. Our data demonstrate that the Mv receptor represents an allelomorphic form of the 'N' antigen on the Ss sialoglycoprotein, rather than variant of the M receptor on the MN sialoglycoprotein. Anti-Mv plus -N (serum Arm.) reacts with the N, 'N' and Mv antigens, whereas anti-Mv (serum Arch.) is specifically directed against the latter receptor.     

Properties and characterization of soluble forms of lymphocyte acetylcholinesterase from an ox

Two soluble forms of AChE from lymphocyte membrane have been obtained, the Triton solubilized Sd form and the high molar salt solubilized Ss form. They present similar Km (0.10 mM). Hydrodynamic properties of these forms have been studied on saccharose gradients with and without detergent or salt. A similar sedimentation coefficient has been found for these two forms (5.7 S). Lymphocyte plasma membrane AChE is a dimeric form (G2). Without detergent, the Sd form shows multiple secondary forms due to main form polymerization. Increase of NaCl concentration (2M) gives rise to a partial dissociation of these polymers. In the same conditions, the Ss form is not affected. The Ss form centrifugated on cesium chloride gradient has a higher density than the Sd form. These two forms have been treated by HPLC: the Stokes radii are respectively 7.1 nm for the Sd form and 4.5 nm for the Ss form. The molecular weights have been estimated at 175 000 for the Sd form and 105 000 for the Ss form. Pronase enzymatic digestion shows that the Ss form is more rapidly inactivated than the Sd form. Phospholipase C inhibits the Ss form and indicates that this form is a lipid-enzyme complex. The Sd form presents a different behaviour: this form is first activated, and afterwards inhibited by phospholipase C. This behaviour could be due to a more preponderant lipidic environment for the Sd form. The Sd form is probably a detergent-lipid-enzyme complex with an important hydrophobocity. These two forms can be explained by a different association between the enzyme and the phospholipids at the plasma membrane.