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Biomedical subjects

I D Walker

Publications and source records attributed to I D Walker.

At least 127 records · Page 7Linked to original sources
PubMed1989

Purification and biochemical characterisation of a novel breast carcinoma associated mucin-like glycoprotein defined by antibody 3E1.2.

A member of the high molecular weight glycoproteins of human milk and breast cancer was isolated from the sera, ascites and breast carcinoma tissue of patients with breast cancer using monoclonal antibody 3E1.2. The 3E1.2 defined antigen, termed mammary serum antigen (MSA) was obtained by immunoaffinity chromatography and a solid phase immuno-precipitation technique (SPIT) from serum of patients with metastatic breast cancer. MSA was found to be a high molecular weight glycoprotein with a Mr greater than 300,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and a native Mr approximately 1 x 10(6) by gel filtration chromatography; in accord with the published Mr of other high molecular weight glycoproteins obtained from human milk and breast cancer. A high degree of glycosylation of MSA molecule was shown by its poor staining with Coomassie blue but good staining in a PAS-silver stain. In addition, MSA contained N-acetyl neuraminic acid and N-acetyl glucosamine as indicated by its binding to wheat-germ agglutinin. The epitope defined by antibody 3E1.2 is sensitive to treatment by sodium periodate and neuraminidase, implying that both carbohydrate and sialic acid are required for binding of antibody 3E1.2. Sandwich immunoassays demonstrated that MSA+ molecules are likely to express repeated 3E1.2 defined epitopes. Furthermore, MSA was susceptible to degradation by pronase, subtilisin and proteinase K and gave a different peptide profile from that of the PAS-O glycoprotein of human milk. MSA+ molecules were found to carry epitopes for a number of other monoclonal antibodies which were reactive with the PAS-O glycoprotein. It is suggested that MSA has the same core protein as is recognised by antibody DF3 which has been used to clone the same cDNA as was cloned with antibodies HMFG-1, HMFG-2 and SM-3. However, the epitope detected by the 3E1.2 antibody is either absent or weakly expressed on human milk, human milk-fat globule membrane (HMFGM) or deglycosylated HMFGM--all of which react strongly with various anti-HMFG antibodies. The antibody 3E1.2 thus recognises a unique epitope of the high molecular weight glycoproteins of human milk and breast cancer, being found in cancer tissue, serum and ascitic fluid of patients with breast cancer but weakly expressed or absent in human milk.

Antibodies, Monoclonal↗
PubMed1989

SP-40,40 is an inhibitor of C5b-6-initiated haemolysis.

This study examines the function of SP-40,40, a newly identified component of the SC5b-9 complement complex, in the regulation of the terminal complement pathway. Purified SP-40,40 was shown to inhibit, in a dose-dependent manner, C5b-6-initiated haemolysis. Apparently additive inhibition was also demonstrated in conjunction with complement S-protein, although SP-40,40 appears to be the more potent inhibitor on an equimolar basis. The data suggest that SP-40,40, like S-protein, probably combines with the nascent C5b-7 complex, forming a cytolytically inactive SC5b-7 - SP-40,40 complex. Preparations of S-protein, purified by an established technique, were shown to be contaminated with SP-40,40. Preparations of affinity-purified SP-40,40 were also shown to contain S-protein, suggesting that these proteins may be partially complexed in plasma.

Animals↗
PubMed1988

The structure of the murine Fc receptor for IgG. Assignment of intrachain disulfide bonds, identification of N-linked glycosylation sites, and evidence for a fourth form of Fc receptor.

The Fc receptor (Fc gamma R) of the murine macrophage cell line, J774, was purified by immunoaffinity chromatography then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequencing. FcR material judged to be pure by these criteria was digested with a number of enzymes to identify the cysteine residues engaged in disulfide bonds within the native structure. The results clearly establish that the mouse macrophage Fc gamma R contains two intrachain disulfide bonds, each of which connects adjacent cysteine residues within the two putative extracellular domains of the molecule. In addition, each disulfide-bonded domain was shown to contain two authentic sites of N-linked glycosylation. Extensive peptide sequencing resulted in the unexpected identification of peptide fragments from a fourth Fc gamma R whose sequences were highly homologous to sequences surrounding the two Cys residues in the amino-terminal domain of both alpha and beta 1 Fc gamma R. The fourth Fc gamma R contains a disulfide-bonded amino-terminal domain similar to beta 1 Fc gamma R.

Amino Acid Sequence↗
PubMed1988

SP-40,40, a newly identified normal human serum protein found in the SC5b-9 complex of complement and in the immune deposits in glomerulonephritis.

We report herein the isolation and initial characterization of a novel protein, termed SP-40,40, which is present at moderate levels (35-105 micrograms/ml) in normal human serum. SP-40,40 is deposited in the renal glomeruli of patients with glomerulonephritis but is not found in normal glomeruli. The protein is a heterodimeric structure of relative molecular mass 80 kD, both chains of which are of a similar size (40 kD). The amino-terminal sequences of both chains are unrelated to one another and possess no significant homology to any known protein sequence. The tissue distribution of SP-40,40 closely resembles that of the terminal complement components and its physicochemical properties are similar to, but distinct from, those of the S protein of complement. We have identified SP-40,40 in the SC5b-9 complex of complement and have demonstrated incorporation of labeled SP-40,40 into this complex. These data suggest that SP-40,40 is an additional component of SC5b-9.

Amino Acid Sequence↗
PubMed1988

Characterization of a 95,000 molecule on sheep leucocytes homologous to murine Pgp-1 and human CD44.

The phagocyte glycoprotein-1 (Pgp-1) antigen of mice is a 94,000 MW molecule with a wide tissue distribution, but no attributed function. We produced a monoclonal antibody (mAb) termed 25-32 which recognizes the Pgp-1 molecule of numerous mammalian species, including humans and sheep. Preclearing experiments with I42/5, a rat anti-mouse Pgp-1 mAb that cross-reacts with human Pgp-1, established the specificity of 25-32 for human and sheep Pgp-1. Moreover, an antibody recognizing human CD44, termed F10-44-2, also reacted with the same molecule as that recognized by 25-32 and I42/5, so establishing the co-identity of CD44 and Pgp-1. Within the sheep thymus, Pgp-1 was expressed most strongly by medullary thymocytes and stromal cells, and by small numbers of cells at the subcapsular cortex. Pgp-1 was expressed early in thymic ontogeny; all 35-40-day gestation fetal sheep thymocytes were intensely Pgp-1+, but by 80 days the number of reactive thymocytes had decreased to adult levels. The expression of Pgp-1 on lymphocytes was markedly increased after stimulation with mitogens, or with phorbol esters and ionomycin. The highly conserved nature of Pgp-1 through evolution, its expression on virtually all cell types within the body, and its increased expression on rapidly dividing cells indicate that this molecule mediates an important function, possibly serving as a hormone or metabolite receptor.

Animals↗
PubMed1987

N-terminal amino acid sequence analyses of the alpha and beta polypeptides from four distinct subsets of sheep major histocompatibility complex class II molecules.

Four monoclonal antibodies, SBU.II 28-1, 37-68, 38-27, and 42-20, each recognizing a distinct, non-overlapping subset of sheep class II molecules, were used to purify class II molecules from a single sheep. Four class II alpha subunits designated 28-1 alpha, 37-68 alpha, 42-20 alpha, and 38-27 alpha and five class II beta subunits designated 28-1 beta, 37-68 beta 1, 37-68 beta 2, 42-20 beta, and 38-27 beta were compared by N-terminal sequence analyses. Two distinct alpha subunits were identified; the 28-1 alpha, 37-68 alpha, and 42-20 alpha subunits all had identical N-terminal amino acids sequences, which exhibited about 75% homology with HLA-DR alpha and mouse E alpha polypeptides. In contrast, the 38-27 alpha sequence exhibited about 80% sequence homology with HLA-DQ alpha and mouse A alpha polypeptides. In general, sheep beta subunits displayed insufficient sequence homology to enable correlation with human beta-chain sequences; however, the 38-27 beta-chain sequence showed homology with the HLA-DQ beta sequence. The conserved sequence surrounding the site for N-linked glycosylation within human/mouse beta polypeptides (residues 19 to 21) was not present in sheep beta sequences and in contrast with the beta-chains of mouse and man, sheep beta polypeptides contained between 1 and 3 positionally variable cysteine residues (residues 13 to 15 inclusive). Individual sheep beta subunits exhibited extensive sequence heterogeneity and each consisted of a unique population of beta polypeptide species. At least 16 different beta polypeptide sequences were identified from a single sheep and the existence of no fewer than nine non-allelic beta genes was inferred from the sequence data. We have previously provided evidence suggesting that the sheep has multiple major histocompatibility complex class II alpha and beta genes related to those of all three HLA-D subregions. The present results suggest that a number of these genes encode HLA-DQ-like heterodimers and that a sheep DR-like alpha gene product is shared with the products of a large and heterogeneous sheep beta gene family.

Amino Acid Sequence↗
PubMed1987

Lethal perinatal osteogenesis imperfecta due to the substitution of arginine for glycine at residue 391 of the alpha 1(I) chain of type I collagen.

A baby with the lethal perinatal form of osteogenesis imperfecta was shown to have a structural defect in the alpha 1(I) chain of type I procollagen. Normal and mutant alpha 1(I) CB8 cyanogen bromide peptides, from the helical part of the alpha 1(I) chains, were purified from bone. Amino acid sequencing of tryptic peptides derived from the mutant alpha 1(I) CB8 peptide showed that the glycine residue at position 391 of the alpha 1(I) chain had been replaced by an arginine residue. This substitution accounted for the more basic charged form of this peptide that was observed on two-dimensional electrophoresis of the collagen peptides obtained from the tissues. The substitution was associated with increased enzymatic hydroxylation of lysine residues in the alpha 1(I) CB8 and the adjoining CB3 peptides but not in the carboxyl-terminal CB6 and CB7 peptides. This finding suggested that the sequence abnormality had interfered with the propagation of the triple helix across the mutant region. The abnormal collagen was not incorporated into the more insoluble fraction of bone collagen. The baby appeared to be heterozygous for the sequence abnormality and as the parents did not show any evidence of the defect it is likely that the baby had a new mutation of one allele of the pro-alpha 1(I) gene. The amino acid substitution could result from a single nucleotide mutation in the codon GGC (glycine) to produce the codon CGC (arginine).

Amino Acid Sequence↗
PubMed1987

Molecular characterization of the murine cytotoxic T-cell membrane glycoprotein Ly-3 (CD8).

The murine Ly-2/3 glycoprotein is a surface marker of T cells restricted by class I major histocompatibility complex antigens. It is a disulfide-bonded heterodimer in which either the alpha or alpha' polypeptide chain encoded by Ly-2 is covalently linked to the beta polypeptide chain encoded by Ly-3. The nucleotide and predicted amino acid sequence of the murine Ly-3 cDNA, isolated by using the rat Ly-3 cDNA clone pX9.15, together with the amino acid sequence of Ly-3.1 peptides and the N terminus, are presented here. The alignment of peptide data from the Ly-3.1 antigen with that of the predicted amino acid sequence of the Ly-3.2 antigen confirmed that the putative Ly-3 cDNA clones do in fact encode the Ly-3 protein. The Ly-3.2 cDNA clones encode a protein of 213 amino acids, which includes a 21-residue leader sequence and structural features in common with immunoglobulin variable, joining, and hinge regions. Searches of protein data bases revealed that Ly-3 is a member of the immunoglobulin superfamily with significant homology to Ly-2, immunoglobulin variable region kappa and lambda light chains, and the beta chain of the T-cell receptor. A single N-linked glycosylation site was found at asparagine-13. The relative expression of two mRNA species (approximately 1.3 and 2.3 kilobases) varied according to the source of mRNA. A murine B1 repeat was located in the 3' untranslated region of Ly-3 cDNA clones.

Amino Acid Sequence↗
PubMed1987

Plasma fibrinolysis during and after normal childbirth.

A detailed sequential study of plasma fibrinolytic components in 42 healthy women during and after childbirth showed that striking changes occurred within minutes of placental separation. Significant increases in overall fibrinolytic activity were detected 15 min after delivery. During this period statistically significant increases in tissue plasminogen activator levels were noted but were overshadowed by a dramatic decrease in the level of plasminogen activator inhibitor (PAI). A 10-fold fall in PAI was noted by days 3-5 post-partum, suggesting that the placenta is the major source of PAI detectable at the end of pregnancy and during labour. Persisting slightly elevated levels of PAI were detected for the first few days post partum. This may be due to increased production of normal 'endothelial' PAI in response to the vascular trauma incurred by placental separation, and in response to uterine vascular changes immediately post-partum.

Antigens↗
PubMed1987

Prophylactic use of antithrombin III concentrate following surgery in congenital antithrombin III deficiency.

Antithrombin III is the major physiological inhibitor of thrombin, and congenital deficiency of antithrombin III is associated with increased risk of venous thrombosis either spontaneously or following trauma, surgery or pregnancy. The successful use of antithrombin III concentrate during and following surgery to prevent venous thrombosis is described in a previously asymptomatic man with familial antithrombin III deficiency.

Antithrombin III↗
PubMed1987

Genetic and biochemical characterization of antigens encoded by the Ly-24 (Pgp-1) locus.

Three allele-specific monoclonal antibodies to Pgp-1 (Ly-24) were used to biochemically characterize the cell surface structures with which they reacted and to map the gene(s) coding for these antigens. The targets of these three monoclonal antibodies (mAb) were shown to be encoded by a gene situated on chromosome 2 close to beta 2m [gene order (Pgp-1-beta 2m-a)] and no recombination between the loci detected by the three antibodies was revealed by genetic analysis. The genetic mapping of loci and tissue distribution of these antigens suggested that they might all correspond to a particular allelic form of the mouse phagocyte glycoprotein-1 (Pgp-1) antigen. Biochemical and serological analysis confirmed that this was indeed the case and revealed that all three mAbs were directed to one epitope. It is surprising that the tissue distribution defined by one mAb (Ly-24A) was different from that for the two other (Ly-24B) antibodies, despite the serological and biochemical identity of their respective targets. The possible reason for this unusual finding is discussed.

Animals↗
PubMed1987

Control of oral anticoagulant treatment by chromogenic prothrombin assay.

Doses of oral anticoagulants in 50 patients on long term treatment were easily and satisfactorily monitored over six months by an automated chromogenic assay of prothrombin (CPA). It is suggested that chromogenic assay of one or more of the vitamin K dependent coagulation factors would provide a readily standardised alternative to those conventional tests which depend on human brain derived reagents, now regarded as a biohazard.

Administration, Oral↗
PubMed1987

Mesenteric venous thrombosis and antithrombin III deficiency.

Of the 123 patients with acute mesenteric infarction treated over the past 12 years, 16 (13%) had mesenteric venous thrombosis. Eight of the patients with mesenteric venous thrombosis survived the initial episode; two have since died. The remaining six patients were studied for evidence of haemostatic deficiencies or abnormalities. Antithrombin III deficiency, which is known to be associated with recurrent venous thrombosis, was found in three patients. It is recommended that all patients with mesenteric venous thrombosis should be screened for antithrombin III deficiency as treatment with coumarin anticoagulants may be indicated, providing effective prophylaxis against further thrombotic episodes.

Adult↗