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1.
Pediatr Cardiol ; 27(3): 309-15, 2006.
Article in English | MEDLINE | ID: mdl-16541219

ABSTRACT

Long-term outcome following the Fontan operation may be affected by the amount of energy lost as blood flows through the anastomosis geometry. A method for detailed quantification of energy loss is applied to computational simulations of the flow in an atriopulmonary and a total cavopulmonary model. Five types of flow (near wall, slow recirculation, medium speed vortices, collision, and streamlined flow) are identified and their energy losses quantified. The presence of recirculation regions decreases the efficiency of the atriopulmonary model, and a region of increased energy loss is seen in the collision region in the total cavopulmonary model. However, the most significant energy loss is through wall shear stress, which is maximal in areas where there is rapid, near wall flow.


Subject(s)
Coronary Circulation , Fontan Procedure , Heart/physiology , Hemorheology , Adult , Anastomosis, Surgical , Blood Flow Velocity , Blood Viscosity , Female , Humans , Magnetic Resonance Imaging , Male , Models, Anatomic , Regional Blood Flow , Stress, Mechanical
2.
Proc Natl Acad Sci U S A ; 98(11): 6279-83, 2001 May 22.
Article in English | MEDLINE | ID: mdl-11353827

ABSTRACT

Polymorphisms in the prion protein gene are known to affect prion disease incubation times and susceptibility in humans and mice. However, studies with inbred lines of mice show that large differences in incubation times occur even with the same amino acid sequence of the prion protein, suggesting that other genes may contribute to the observed variation. To identify these loci we analyzed 1,009 animals from an F2 intercross between two strains of mice, CAST/Ei and NZW/OlaHSd, with significantly different incubation periods when challenged with RML scrapie prions. Interval mapping identified three highly significantly linked regions on chromosomes 2, 11, and 12; composite interval mapping suggests that each of these regions includes multiple linked quantitative trait loci. Suggestive evidence for linkage was obtained on chromosomes 6 and 7. The sequence conservation between the mouse and human genome suggests that identification of mouse prion susceptibility alleles may have direct relevance to understanding human susceptibility to bovine spongiform encephalopathy (BSE) infection, as well as identifying key factors in the molecular pathways of prion pathogenesis. However, the demonstration of other major genetic effects on incubation period suggests the need for extreme caution in interpreting estimates of variant Creutzfeldt-Jakob disease epidemic size utilizing existing epidemiological models.


Subject(s)
Genetic Linkage , Prion Diseases/genetics , Quantitative Trait, Heritable , Animals , Chromosome Mapping , Female , Male , Mice , Time Factors
3.
Br J Haematol ; 108(2): 448-54, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10691880

ABSTRACT

Duffy blood group antigens are carried on a glycoprotein that is predicted to pass through the erythrocyte membrane seven times and is a promiscuous chemokine receptor. The Fy(a- b-) phenotype is present in two-thirds of African-American Blacks but is rare in Caucasians. In Blacks, the phenotype is due to a non-functional GATA-1 motif in the FY B, which silences the gene in erythrocytes but not in other tissues, and these patients do not generally make anti-Fyb or anti-Fy3. We describe here the molecular analysis of FY in three unrelated Caucasians who were studied because they had strong anti-Fy3 in their serum. Each was found to have a point mutation that was predicted to change a tryptophan to a premature stop codon in the coding sequence. In one patient (patient 1), the nonsense mutation was at nucleotide 287 of the major transcript in FY A; in another (patient 2), it was at nucleotide 407 in the major transcript of FY B; and in a third (patient 3), it was at nucleotide 408 of the major transcript of FY A.


Subject(s)
Duffy Blood-Group System/genetics , Point Mutation/genetics , Aged , Aged, 80 and over , Codon, Nonsense/genetics , Codon, Terminator/genetics , Fatal Outcome , Female , Genotype , Humans , Male , Middle Aged , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length
4.
Transfus Med ; 8(2): 141-7, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9675792

ABSTRACT

Decay-accelerating factor (DAF, CD55) is a complement regulatory glycoprotein that expresses the Cromer-system blood group antigens. Two, very rare, inherited DAF-deficiency phenotypes, Inab and Dr(a-), were identified in Japanese propositi. Red cells of the Inab phenotype propositus had no Cromer-system antigens and did not bind monoclonal anti-DAF. The Inab propositus was homozygous for a DAF non-sense mutation, converting the Trp53 codon to a stop codon; her parents were heterozygous for this mutation. This is the same mutation as that previously found in the original Inab phenotype propositus. Haemagglutination-inhibition titrations of the serum of the Inab propositus with soluble-recombinant DAF demonstrated that anti-IFC represents a mixture of antibodies to all four DAF short consensus repeat domains. The Dr(a-) individual had very low levels of Cromer-system antigens and DAF on her red cells. Loss of a TaqI restriction site from DAF exon 5 suggested that she has a previously detected mutation, encoding a Ser165Leu substitution. Red cells of the two propositi did not show abnormal levels of lysis in an acid lysis test, but after blocking of CD59 with monoclonal antibody, Inab phenotype red cells showed more lysis than Dr(a-) red cells, and Dr(a-) cells showed substantially more lysis than control cells.


Subject(s)
Blood Group Antigens/genetics , CD55 Antigens/genetics , Erythrocytes/immunology , Mutation , Adult , Blood Group Antigens/immunology , CD55 Antigens/immunology , Child , Female , Heterozygote , Homozygote , Humans , Male , Middle Aged , Phenotype
5.
Br J Haematol ; 103(4): 1184-91, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9886340

ABSTRACT

The molecular basis of the three major alleles (Fy(a)/Fy(b)/Fy) of the Duffy (FY) blood group system has recently been established but the Fy(x) phenotype associated with weak expression of the Fy(b) and other FY antigens is poorly understood. In the Fy(x) genes of five unrelated British and Swedish donors with the Fy(a+b+weak) phenotype we found two missense mutations predicting amino acid changes Arg89Cys and Ala100Thr in the FY glycoprotein. The same mutations were found in two Fy(a-b+weak) samples from individuals of Swedish and Algerian origin. Their red blood cells showed a marked decrease in Fy(b), Fy3 and Fy6 expression measured by routine serology and flow cytometry. The rare FY genotypes Fy(x)Fy(x) and Fy(x)Fy were confirmed by family studies and DNA sequencing. Screening by allele-specific primer PCR (ASP-PCR) for these mutations among 100 Caucasian and 100 Black random blood donors indicated allele frequencies of 2.5% and 0% respectively. Ala100Thr alone was present in 33% of the Caucasians (but none of the Blacks) with no weakening of FY expression. A novel allele at the FY locus associated with the Fy(x) phenotype was studied. Mistyping of this weak Fy(b) antigen in clinical transfusion medicine may lead to delayed haemolytic transfusion reactions in immunized patients. A potential role for genomic typing is proposed.


Subject(s)
Duffy Blood-Group System/genetics , Mutation, Missense/genetics , Alleles , Flow Cytometry , Humans , Phenotype , Polymerase Chain Reaction/methods , Sequence Analysis, DNA
6.
Blood ; 89(11): 4219-25, 1997 Jun 01.
Article in English | MEDLINE | ID: mdl-9166867

ABSTRACT

Lutheran glycoprotein (Lu gp) has five predicted immunoglobulin superfamily (IgSF) domains. K562 cells were transfected with Lu cDNA and tested by flow cytometry with monoclonal antibodies and Lu blood group antisera. The results confirmed the identity of Lu cDNA. Deletion mutants lacking the regions encoding one or more IgSF domains were made by inverse polymerase chain reaction (PCR), expressed in K562 cells, and tested with the same antibodies. The Lu(b) and Lu5 antigens and the epitope recognized by monoclonal antibody BRIC 224 were mapped to the first, N-terminal, IgSF domain. Lu4 and Lu8 were mapped to domain 2; Lu20 to domain 3; Lu7 and BRIC 221 epitope to domain 4, and Lu13 and Au(b) to domain 5. The organization of the LU gene was determined. The region encoding the open reading frame is arranged in 15 exons extending over approximately 11 kb on chromosome 19q13.2. The Lu(a)/Lu(b) and Au(a)/Au(b) blood group polymorphisms were studied using genomic DNA from typed blood donors. The Lu(a) mutation is a base change in exon 3 (G252 to A) encoding an Arg77 (Lu(b)) to His (Lu(a)) change on the CFG face of domain 1. The Au(a)/Au(b) polymorphism is an A1637 to G substitution in exon 12 encoding a Thr539 (Au(a)) to Ala (Au(b)) change on the G strand of domain 5.


Subject(s)
Chromosomes, Human, Pair 19 , Genes, Immunoglobulin , Immunoglobulins/genetics , Lutheran Blood-Group System/genetics , Cell Line , Chromosome Mapping , Epitope Mapping , Gene Deletion , Humans , Polymorphism, Genetic
7.
Br J Haematol ; 90(4): 823-9, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7669660

ABSTRACT

The erythrocyte chemokine receptor, a receptor for Plasmodium vivax, carries the antigens of the Duffy blood group system. Sequence analysis of reticulocyte RNA from individuals of known Duffy phenotype showed that the Fya antigen differs from the Fyb antigen as a result of a single nucleotide difference (A131 or G) encoding amino acid Gly44 (Fya) or Asp (Fyb) in the N-terminal extracellular domain of the glycoprotein. Evidence is presented for two different genetic backgrounds giving rise to the Fy(a-b-) phenotype. The most likely genetic mechanism in most individuals of the Fy(a-b-) phenotype is down-regulation of Duffy glycoprotein mRNA. However, the Duffy gene from a very rare Caucasian individual (AZ) with the Fy(a-b-) phenotype has a 14 base-pair deletion (nucleotides 287-301) resulting in a frameshift which introduces a stop codon and produces a putative truncated 118 amino acid protein. The occurrence of this mutation in an apparently healthy individual raises questions about the functional importance of the Duffy glycoprotein not only in normal erythrocytes but also in all human cells and tissues.


Subject(s)
Duffy Blood-Group System/genetics , Erythrocytes/physiology , Gene Deletion , Membrane Glycoproteins/genetics , Receptors, Cytokine/genetics , Amino Acid Sequence , Base Sequence , Humans , Molecular Sequence Data , Phenotype , Polymerase Chain Reaction , Polymorphism, Genetic , Polymorphism, Restriction Fragment Length , Receptors, Cell Surface , Sequence Deletion
8.
Proc Natl Acad Sci U S A ; 92(12): 5496-500, 1995 Jun 06.
Article in English | MEDLINE | ID: mdl-7777537

ABSTRACT

Glycoproteins expressing the Lutheran blood group antigens were isolated from human erythrocyte membranes and from human fetal liver. Amino acid sequence analyses allowed the design of redundant oligonucleotides that were used to generate a 459-bp, sequence-specific probe by PCR. A cDNA clone of 2400 bp was isolated from a human placental lambda gt 11 library and sequenced, and the deduced amino acid sequence was studied. The predicted mature protein is a type I membrane protein of 597 amino acids with five potential N-glycosylation sites. There are five disulfide-bonded, extracellular, immunoglobulin superfamily domains (two variable-region set and three constant-region set), a single hydrophobic, membrane-spanning domain, and a cytoplasmic domain of 59 residues. The overall structure is similar to that of the human tumor marker MUC 18 and the chicken neural adhesion molecule SC1. The extracellular domains and cytoplasmic domain contain consensus motifs for the binding of integrin and Src homology 3 domains, respectively, suggesting possible receptor and signal-transduction function. Immunostaining of human tissues demonstrated a wide distribution and provided evidence that the glycoprotein is under developmental control in liver and may also be regulated during differentiation in other tissues.


Subject(s)
Gene Expression Regulation, Developmental , Liver/metabolism , Lutheran Blood-Group System/genetics , Amino Acid Sequence , Antibodies, Monoclonal , Base Sequence , Cell Membrane/metabolism , Chromosome Mapping , Chromosomes, Human, Pair 19 , DNA, Complementary , Erythrocytes/metabolism , Genes, Immunoglobulin , Humans , Immunohistochemistry , Liver/embryology , Lutheran Blood-Group System/chemistry , Molecular Sequence Data , Trophoblasts/metabolism
9.
Blood ; 84(4): 1276-82, 1994 Aug 15.
Article in English | MEDLINE | ID: mdl-7519480

ABSTRACT

The human erythrocyte blood group system Cromer consists of high-incidence and low-incidence antigens that reside on decay-accelerating factor (DAF; CD55), a glycosyl-phosphatidylinositol-anchored membrane protein that regulates complement activation on cell surfaces. In the Cromer phenotypes Dr(a-) and Inab there is reduced or absent expression of DAF, respectively. This study investigated the molecular basis of the reduced DAF expression by polymerase chain reaction amplification of genomic DNA and RNA/cDNA obtained from Epstein-Barr virus-transformed lymphoblastoid cell lines. Sequence analysis of the Inab propositus showed a single nucleotide substitution in exon 2 of the DAF gene and at the corresponding position in the cDNA, G314-->A resulting in Trp53-->Stop. This truncation near the amino terminus explains the complete absence of surface DAF in the Inab phenotype. A similar analysis was performed for two Dr(a-) individuals, including KZ, who was previously reported to be Inab phenotype but is now shown by immunochemical and serologic methods to be Dr(a-) phenotype. A single nucleotide change was found in exon 5 of the DAF gene, C649-->T resulting in Ser165-->Leu, which we had previously shown to lead to loss of the Dra epitope. However, two species of cDNA were found, one encoding full-length DAF with the single amino acid change and the more abundant species having a 44-nucleotide deletion. The 44 nucleotide deletion includes the single polymorphic site, which creates a cryptic branch point in the Dr(a-) allele that leads to use of a downstream cryptic acceptor splice site. This shifts the reading frame and leads to a premature stop codon that precludes membrane anchoring. Thus, the single point mutation in the Dr(a-) phenotype results in a novel use of alternative splicing and provides a molecular explanation for both the antigenicity and the reduced DAF expression seen in this phenotype.


Subject(s)
Antigens, CD/biosynthesis , Blood Group Antigens/genetics , Erythrocytes/metabolism , Membrane Glycoproteins/biosynthesis , Point Mutation , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Base Sequence , Blood Proteins/biosynthesis , CD55 Antigens , Cell Line, Transformed , Consensus Sequence , Gene Expression , Herpesvirus 4, Human/genetics , Humans , Lymphocytes , Mice/immunology , Molecular Sequence Data , Phenotype , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Restriction Mapping
11.
Blood ; 82(10): 3198-203, 1993 Nov 15.
Article in English | MEDLINE | ID: mdl-8219208

ABSTRACT

Glycophorin C (GPC) and glycophorin D (GPD) are homologous sialoglycoproteins in the human red blood cell membrane. Both are thought to be encoded by the GPC gene (GYPC). We report that the rare blood group antigen, Ana, is expressed on GPD but not on GPC. cDNA was synthesized from total RNA obtained from two unrelated, heterozygous Ana+ blood donors and analyzed by the polymerase chain reaction using primers that spanned sequences encoded by the GYPC gene. The expected 412-bp fragment was generated, and sequencing of the amplified product showed a G-->T substitution at nucleotide 67 of the coding sequence, resulting in the substitution of alanine by serine at amino acid residue 23 of GPC and, presumably, residue 2 of GPD. To explain the expression of Ana on GPD but not on GPC, we postulate that the conformation of the amino acid residues at the N-terminal region of GPD determines the antigenic expression as this conformation would be different from that of the same sequence of amino acids occurring within GPC. Other possible reasons for antigen expression on a shorter protein product but not on the full-length protein product of the same gene are discussed. We extrapolate this reasoning to account for the expression of the common GE2 blood group antigen on GPD but not on GPC.


Subject(s)
Blood Group Antigens/immunology , Glycophorins/genetics , Point Mutation , Amino Acid Sequence , Base Sequence , DNA, Complementary/chemistry , Glycophorins/immunology , Humans , Molecular Sequence Data
12.
J Biol Chem ; 267(21): 15134-9, 1992 Jul 25.
Article in English | MEDLINE | ID: mdl-1634548

ABSTRACT

We have raised a rabbit antiserum to a synthetic peptide corresponding to the C terminus (residues 400-416) of the Rh30A polypeptide. The rabbit antiserum reacted with the Rh30B (D30) polypeptide in addition to the Rh30A (C/c and/or E/e) polypeptide(s), indicating that these proteins share homology at their C termini. The antiserum did not react with erythrocyte membranes from an individual with Rh(null) syndrome. The rabbit antiserum immunoprecipitated Rh polypeptides from erythrocyte membranes and alkali-stripped membranes, but not from intact erythrocytes. Treatment of intact red cells with carboxypeptidase Y did not affect the reactivity of the antiserum, whereas treatment of alkali-stripped and unsealed erythrocyte ghost membranes resulted in the loss of antibody binding. Carboxypeptidase A treatment of intact erythrocytes and alkali-stripped membranes had no effect on antibody binding, indicating that the C-terminal domains of the Rh polypeptides contain lysine, arginine, proline, or histidine residues. These results show that the C termini of the Rh polypeptides are located toward the cytoplasmic face of the erythrocyte membrane. Treatment of intact radioiodinated erythrocytes with bromelain followed by immunoprecipitation with monoclonal anti-D gave a band of M(r) 24,000-25,000, indicating that the Rh30B (D30) polypeptide is cleaved at an extracellular domain close to the N or C terminus, with loss of the major radioiodinated domain. Immunoblotting of bromelain treated D-positive erythrocyte membranes with the rabbit antiserum to the C-terminal peptide revealed a new band of M(r) 6000-6500, indicating that the extracellular bromelain cleavage site is located near the C terminus of the molecule. The band of M(r) 6000-6500 was not obtained in erythrocyte membranes derived from bromelain treated D-negative erythrocytes. Erythrocytes of the rare -D- phenotype appear to either totally lack, or have gross alterations in, the Cc/Ee polypeptide(s), since the bromelain treatment of these cells resulted in the total loss of staining in the M(r) 35,000-37,000 region and the concomitant appearance of the new band of M(r) 6000-6500.


Subject(s)
Erythrocyte Membrane/metabolism , Peptides/metabolism , Amino Acid Sequence , Blotting, Western , Carboxypeptidases , Carboxypeptidases A , Electrophoresis, Polyacrylamide Gel , Humans , Immune Sera , Molecular Sequence Data , Precipitin Tests
13.
Immunology ; 75(3): 507-12, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1374058

ABSTRACT

CD59 is a widely expressed cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein which acts as an inhibitor of the assembly of the membrane attack complex of autologous complement. Four new monoclonal antibodies to CD59 (2/24, 1B2, BRIC 229, BRIC 257) are described. Competitive binding experiments using these antibodies, two known CD59 antibodies (MEM-43, YTH 53.1) and a previously described antibody LICR-LON-Fib75.1 demonstrated that all seven antibodies see related epitopes on human erythrocyte CD59. In common with other GPI-linked proteins, CD59 (as defined by antibody 2/24) was sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) on lymphocytes and monocytes but not on erythrocytes. Flow cytometric analysis using antibody 2/24 identified two populations (CD59 positive and CD59 deficient) of lymphocytes, monocytes and erythrocytes in peripheral blood from a patient with paroxysmal nocturnal haemoglobinuria (PNH). The abundance of CD59 on normal erythrocytes was determined as 21,000 copies/cell when radioiodinated BRIC 229 was used. Other CD59 antibodies gave values of 10,000 (IF5) and 15,000 (2/24) against the same target cells. Radioiodinated Fab fragments of BRIC 229 gave a value of 39,000 copies/cell. Erythrocytes from two individuals with a rare inherited deficiency of decay accelerating factor (DAF), known as the Inab phenotype, expressed normal levels of CD59.


Subject(s)
Antibodies, Monoclonal/immunology , Antigens, CD/immunology , Erythrocytes/immunology , Hemoglobinuria, Paroxysmal/immunology , Membrane Glycoproteins/immunology , Membrane Proteins/deficiency , Antigens, CD/analysis , CD55 Antigens , CD59 Antigens , Cell Line , Epitopes/drug effects , Humans , Immunoblotting , Membrane Glycoproteins/analysis , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , Phosphoric Diester Hydrolases/pharmacology
14.
Vox Sang ; 63(1): 56-8, 1992.
Article in English | MEDLINE | ID: mdl-1413665

ABSTRACT

The blood group Duch (Dha) antigen is located on glycophorin C (GPC). Total RNA prepared from the reticulocyte fraction of two Dh(a+) individuals were used in the synthesis of first-strand cDNA. The first-strand cDNA served as templates for the amplification of GPC-related DNA by polymerase chain reaction (PCR). The expected PCR product consisted of 412 base pairs. On sequencing the PCR-amplified DNA, a base change (cytosine----thymidine) at nucleotide 40 of the GPC cDNA was detected. Thus, the variant GPC (GPC.Dha) on Dh(a+) red cells has a substitution of leucine by phenylalanine at amino acid residue 14.


Subject(s)
Blood Group Antigens/genetics , Glycophorins/genetics , Base Sequence , Gene Expression Regulation , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed
15.
Blood ; 78(12): 3291-7, 1991 Dec 15.
Article in English | MEDLINE | ID: mdl-1720702

ABSTRACT

A 38-year-old Russian woman (KZ) has been identified as the fourth proposita with the Inab blood group phenotype. Like the first two propositi, she has a chronic intestinal disorder and, as shown for the third proposita, her Inab phenotype is demonstrably inherited. KZ's serum contained anti-IFC, which reacted with a red blood cell (RBC) membrane component with an Mr of 70,000, which is decay accelerating factor (DAF). Her RBCs lacked all Cromer-related blood group antigens and DAF. Her RBCs were no more susceptible than normal control RBCs to lysis in acid lysis or in rabbit or human antibody-initiated complement lysis tests. Northern blots of total RNA isolated from KZ's Epstein-Barr virus-transformed lymphoblasts showed a marked reduction of DAF mRNA when compared with normal. Polymerase chain reaction (PCR) amplification of cDNA confirmed this reduced level of DAF mRNA. Sequencing of the PCR product showed a 44-nucleotide deletion in the mRNA close to the short consensus repeats IIIa/IIIb intron/exon boundary. This deletion results in a change in the reading frame that places a termination codon six amino acids after the deletion. The putative translation product would lack a glycosyl phosphatidyl-inositol linkage site and, therefore, would not be membrane-bound in the RBC.


Subject(s)
Blood Group Antigens/genetics , Complement Inactivator Proteins/deficiency , Erythrocytes/immunology , Membrane Proteins/deficiency , Phenotype , Adult , Amino Acid Sequence , Antibodies/metabolism , Base Sequence , Blood Group Antigens/immunology , Blotting, Northern , CD55 Antigens , Complement C3/metabolism , Complement Inactivator Proteins/genetics , DNA/chemistry , Erythrocytes/chemistry , Erythrocytes/physiology , Female , Hemolysis , Humans , Immunoblotting , Membrane Proteins/genetics , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/chemistry
16.
Immunology ; 74(2): 197-205, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1721039

ABSTRACT

The cell-surface glycoproteins CD44 and CD58 are involved in cell adhesion reactions. In this paper 12 monoclonal antibodies in CD44 and two in CD58 are described. Competitive binding assays using CD44 antibodies identified three distinct epitope groups. Antibodies in Group 1 and, with one exception (BRIC 214), antibodies in group 2, but not antibodies in Group 3, recognized epitopes that are sensitive to reduction and to trypsin or chymotrypsin treatment of intact erythrocytes, and so these epitopes probably reside on the N-terminal disulphide-bonded domain of CD44. Antibodies in CD44 did not inhibit the binding of CD58 antibodies to erythrocytes or vice versa. Quantitative binding studies using radioiodinated IgG measured 1888-5592 copies of CD44 and 1772-3290 copies of CD58 on normal erythrocytes. Similar measurements with radioiodinated Fab fragments gave values of 6508-10,450 (CD44) and 3457-7622 (CD58). Immunocytochemical studies indicated that CD44 is much more widely expressed in non-haemopoietic tissues than CD58. Comparison with previously described CD44 antibodies suggests that antibodies in our Group 1 encompass Hermes 2 and that those in Group 2 encompass Hermes 1. All the CD44 antibodies gave weakened reactions with Lu(a-b-) erythrocytes of the In(Lu) type by one or more methods. BRIC 214 and antibodies in epitope Group 3 were used to demonstrate that CD44 on these variant cells gives membrane-bound trypsin and chymotrypsin cleavage fragments of similar molecular weight to those obtained with normal erythrocytes.


Subject(s)
Antigens, Surface/analysis , Erythrocytes/immunology , Membrane Glycoproteins/analysis , Receptors, Lymphocyte Homing/analysis , Antibodies, Monoclonal/immunology , Binding, Competitive/immunology , CD58 Antigens , Epitopes/analysis , Humans , Immunoblotting , Tissue Distribution
17.
Transfusion ; 30(3): 222-5, 1990.
Article in English | MEDLINE | ID: mdl-2107609

ABSTRACT

The human red cell membrane components reacting with monoclonal antibody MB-2D10 were examined by immunoblotting. The antibody bound to a diffusely staining band extending from Mr 30,000 up to the high-molecular-weight region of the gel in normal membranes and in Rhnull U + membranes, but not in Rhnull U - membranes. Treatment of normal red cells with an endoglycosidase F-containing preparation destroyed the epitope recognized by MB-2D10. The reactivity of the antibody with purified preparations of Rh-related glycoproteins D30 polypeptide, D50 polypeptide, R6A32 polypeptide, and R6A45 polypeptide was also examined. Only the purified R6A45 and D50 components reacted with MB-2D10. These results show that MB-2D10 recognizes a carbohydrate-dependent epitope on the R6A45 and D50 group of Rh-related polypeptides. The results also suggest the possibility that the U antigen arises from interaction between glycophorin B and the Rh-related components D50 and R6A45.


Subject(s)
Antibodies, Monoclonal/immunology , Erythrocyte Membrane/analysis , Glycoproteins/blood , Rh-Hr Blood-Group System , Animals , Humans , Immunoblotting , Mice
18.
Biochem J ; 261(2): 489-93, 1989 Jul 15.
Article in English | MEDLINE | ID: mdl-2476116

ABSTRACT

1. We have studied the peripheral blood cells of an individual with the Inab phenotype who is deficient in decay accelerating factor (DAF). 2. In contrast with the situation in paroxysmal nocturnal haemoglobinuria, membranes from peripheral blood cells of the Inab phenotype individual lack DAF, but retain the other glycosylphosphatidylinositol-linked proteins acetylcholinesterase and LFA-3. 3. Unlike normal Epstein-Barr-virus-transformed lymphoblastoid cell lines (EBV-LCL), DAF was not expressed on EBV-LCL derived from peripheral blood lymphocytes of the Inab individual. 4. No differences in the DAF gene of normal and Inab phenotype individuals could be detected by Southern blotting studies. 5. EBV-LCL derived from the Inab individual had a gross reduction in the level of DAF mRNA compared with normal EBV-LCL. 6. Our results suggest that the DAF gene in the Inab phenotype contains a mutation which affects the transcription or processing of DAF mRNA.


Subject(s)
Blood Cells/analysis , Blood Proteins/deficiency , Complement Inactivator Proteins/deficiency , Membrane Proteins/deficiency , Blood Proteins/genetics , CD55 Antigens , Complement Inactivator Proteins/blood , Complement Inactivator Proteins/genetics , Erythrocytes/analysis , Humans , Membrane Proteins/blood , Membrane Proteins/genetics , Phenotype , RNA, Messenger/analysis
19.
Nurs Times ; 85(9): 31-4, 1989.
Article in English | MEDLINE | ID: mdl-2928252
20.
Carbohydr Res ; 178: 203-12, 1988 Jul 15.
Article in English | MEDLINE | ID: mdl-3142686

ABSTRACT

Endo-N-acetyl-beta-D-glucosaminidase F-Peptidyl N-glycosidase F preparations (abbreviated Endo F) and endo-beta-D-galactosidase were used to study the major human erythrocyte membrane glycoproteins and the components carrying the blood group A, B, Rhesus (D), and Duffy (Fya) antigens. The results are consistent with the known presence of an N-glycosyl-linked oligosaccharide on sialoglycoprotein alpha and the absence of such an oligosaccharide from sialoglycoprotein delta. Under the conditions used, only a portion of the N-glycosyl-linked oligosaccharides on band 3 molecules were cleaved by Endo F alone or by Endo F in combination with endo-beta-D-galactosidase. Immunoblotting experiments showed that treatment of red cells with Endo F alone had little effect on the components carrying blood group A and B antigen activity. However, Endo F used in combination with endo-beta-D-galactosidase caused a substantial reduction in the binding of monoclonal anti-A and anti-B antibodies. The results clearly show that sialoglycoproteins alpha and delta carry little or no blood group A or B activity. Endo F alone, or in combination with endo-beta-D-galactosidase, had no effect on the electrophoretic mobility of the Rh(D) polypeptide, supporting previous suggestions that this membrane polypeptide is unusual in not being glycosylated. Endo F had a dramatic effect on the electrophoretic mobility of the component(s) carrying blood group Fya activity. The diffuse Fya component of Mr 38,500-90,000 was sharpened to a band of Mr 26,000. Either endo-beta-D-galactosidase or neuraminidase treatment reduced the Mr of the Fya component(s) but did not significantly sharpen the bands, suggesting that the Fya component contains between 40-50% by mass of N-glycosyl-linked oligosaccharides.


Subject(s)
Erythrocytes/metabolism , Glycoside Hydrolases/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase , Sialoglycoproteins/blood , Surface Properties
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