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M C Owen

Publications and source records attributed to M C Owen.

At least 19 recordsLinked to original sources
PubMed2002

Macro-alkaline phosphatase due to IgG kappa complex: demonstration with polyethylene glycol precipitation and immunofixation.

An otherwise unexplained, persistently elevated plasma alkaline phosphatase concentration in a 71-year-old woman was found to be attributable to the presence of macro-alkaline phosphatase using polyethylene glycol precipitation. Gel filtration showed two high MW peaks with masses of about 330 kDa and 560 kDa. The alkaline phosphatase (ALP) complex was characterized by immunoelectrophoresis as a complex with IgG with kappa light chains.

Aged↗
PubMed2002

Prostate specific antigen inhibits immune responses in vitro: a potential role in prostate cancer.

PURPOSE: Prostate specific antigen (PSA) is found in high concentration in prostate tissue and in semen, in which its physiological function appears to be liquefaction. In prostate cancer the peripheral PSA concentration is elevated, which may be used as a disease marker. Systemic and local immune defects have been demonstrated in prostate cancer and we postulated a role for PSA in this immunosuppression. We explored the effects of PSA on human T-lymphocyte proliferation in vitro. MATERIALS AND METHODS: PSA was purified from normal seminal plasma using a modified chromatographic technique. The effect of PSA or control protein on lymphocyte responses to mitogens, tetanus toxoid and alloantigens was tested. The inhibitory effect observed was further explored by varying the time of PSA addition, denaturing PSA and including interleukin-2 and anti-PSA antibodies. RESULTS: PSA suppressed in vitro phytohemagglutinin and alloantigen stimulated lymphocyte proliferation in a dose dependent manner. This effect was reversed by adding anti-PSA antibodies but not by interleukin-2. CONCLUSIONS: These in vitro PSA effects suggest another T-lymphocyte mediated immunosuppressive mechanism. In vivo high levels of PSA may compromise natural immune responses to cancer and current attempts at immunotherapy for prostate cancer.

Disease Progression↗
PubMed1994

Hb Footscray or alpha 133(H16) Ser-->Arg: a new hemoglobin variant.

Hb Footscray, alpha 133(H16) Ser-->Arg, is a newly described hemoglobin variant found in an adult male of Polish-Hungarian descent. Hematological data and stability by the isopropanol stability test were normal. The abnormal hemoglobin comprised 15% of total hemoglobin and migrated as a split band in the Hb F position on cellulose acetate at pH 8.6. Like Hb Manitoba, which is also a Ser-->Arg mutation and occurs in a very similar spatial position, the split bands presumably arise from the formation of asymmetric hybrids between Hb Footscray and Hb A.

Adult↗
PubMed1993

Hb Howick [beta 37(C3)Trp-->Gly]: a new high oxygen affinity variant of the alpha 1 beta 2 contact.

Hb Howick or beta 37(C3) Trp-->Gly is a newly described hemoglobin variant found in an adult male. Hematological data and stability by the isopropanol stability test were normal. The abnormal variant comprised 29% of total hemoglobin and migrated in the Hb D position on cellulose acetate at pH 8.6, and in the Hb F position on citrate agar (pH 6.0). Oxygen dissociation studies on the whole blood showed the variant to have a higher oxygen affinity than normal, with a P50 of 19.8 mm Hg (normal, 26 mm Hg). There were also significant differences in the saturation curve. The variant showed a reduced Bohr effect which was manifested as very high oxygen affinity at low pH and saturation. The beta 37 residue is an alpha 1 beta 2 contact site and the substitution of the tryptophan for a glycine would be expected to result in a destabilization of the deoxy-hemoglobin form because of the reduced number of hydrogen bonds, salt bridges and van der Waal contacts between the alpha 1 and beta 2 chains.

Adult↗
PubMed1992

A competitive ELISA for lipoprotein(a).

A competitive ELISA for lipoprotein(a) (Lp(a)) is described. The method uses a commercially available polyclonal anti-Lp(a) antibody and an IgG biotinstreptavidin-horseradish peroxidase detection system. The method is simple and robust with an assay sensitivity of 0.7 ng/well (1.4 micrograms/l). The antibody cross-reactivity was 0.14% against LDL and 0.70% against plasminogen. The coefficients of variation obtained with control sera of 266 and 552 mg/l were: 5.0% and 4.6% (n = 6), respectively for the intraassay; and 10.8% and 9.5% (n = 16), respectively for the interassay. The method showed an excellent correlation with a commercial immunoradiometric assay (IRMA), y (ELISA) = 0.94x (IRMA) - 8, (r = 0.98). A recovery study in which a 200 mg/L standard and four plasma samples were diluted with different proportions of a low plasma sample, gave linear relationships and also confirmed the specificity of the antibody.

Binding, Competitive↗
PubMed1991

P1 variant antithrombins Glasgow (393 Arg to His) and Pescara (393 Arg to Pro) have increased heparin affinity and are resistant to catalytic cleavage by elastase. Implications for the heparin activation mechanism.

The heparin affinity of normal and two P1 variants of antithrombin-III (AT) was studied by gradient elution with NaCl in Tris buffer on heparin-Sepharose. At pH 7.4 normal AT eluted at [Na+] 0.78 mol/l and the variants both showed increased affinity with AT Pescara eluting at [Na+] 0.86 mol/l and AT Glasgow at [Na+] 0.92 mol/l. We have earlier proposed a model for heparin activation in which the native state of AT maintains a salt bridge involving the P1 Arg-393 residue. Binding of heparin induces a higher heparin affinity conformation in which the salt bridge is disrupted to reveal the reactive centre for inhibition of thrombin. The Glasgow and Pescara variants, lacking a reactive centre P1 basic residue, would be unable to form this salt bridge, and we suggested that the high affinity conformation which they adopt as their native state would resemble the heparin induced conformation. To examine this model, we measured the heparin induced fluorescence of two P1 variants and tested the susceptibility of their reactive loops to catalytic cleavage. Both variants had fluorescence spectra indistinguishable from normal AT. In the absence of heparin, neither variant was more susceptible than normal to catalytic cleavage by human neutrophil elastase. These findings suggest that the conformation of these P1 variants is different to that of fully heparinized normal AT.

Antithrombin III↗
PubMed1990

Antithrombin III.

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Amino Acid Sequence↗
PubMed1989

Conformational lability of vitronectin: induction of an antigenic change by alpha-thrombin-serpin complexes and by proteolytically modified thrombin.

We previously showed that the alpha-thrombin-antithrombin III complex causes antigenic change in vitronectin as monitored by the monoclonal anti-vitronectin antibody 8E6 (Tomasini & Mosher, 1988). We have extended these studies to other protease-serpin complexes and to gamma-thrombin, a proteolytic derivative of alpha-thrombin. In the presence of heparin, recognition of vitronectin by 8E6 was increased 64- or 52-fold by interaction with the complex of alpha-thrombin and heparin cofactor II or the Pittsburgh mutant (Met358----Arg) of alpha 1-protease inhibitor, respectively. This was comparable to the value obtained with the alpha-thrombin-antithrombin III complex. Factor Xa-serpin complexes were approximately 4-fold less effective than the corresponding thrombin complexes. alpha-Thrombin-serpin complexes but not Xa-serpin complexes formed disulfide-bonded complexes with vitronectin. Antigenic changes and disulfide-bonded complexes were not detected when trypsin- or chymotrypsin-serpin complexes were incubated with vitronectin. gamma-Thrombin caused 7- and 34-fold increases in recognition of vitronectin by MaVN 8E6 in the absence and presence of heparin, respectively. In contrast, alpha-thrombin by itself had no effect. The antigenic change induced by gamma-thrombin was maximal when gamma-thrombin and vitronectin were equimolar, was not dependent on cleavage of vitronectin, and was abolished by inhibition of gamma-thrombin with Phe-Pro-Arg-chloromethyl ketone but not with diisopropyl fluorophosphate. These data indicate that alpha-thrombin is the component in alpha-thrombin-serpin complexes that induces the antigenic change in vitronectin, probably via a region that is preferentially exposed in gamma-thrombin.

Animals↗
PubMed1989

Molecular characterization of antithrombin Barcelona-2: 47 arginine to cysteine.

The molecular characterization of antithrombin Barcelona-2 is reported. The abnormal antithrombin was isolated from plasma by chromatography on heparin-Sepharose at pH 6.0, and ion exchange on DEAE-Sephadex at pH 8.6 and 6.0. The tryptic peptides were mapped by reverse-phase HPLC and amino acid sequencing and mass spectrometry showed arginine-47 to be replaced by cysteine. The affinity of Barcelona-2 for heparin is dramatically decreased. The new cysteine does not form a mixed disulphide with DTNB, implying it is present as a disulphide with some other available thiol molecule such as cysteine. This extra bulk at position 47 accounts for the low heparin affinity compared with two other mutations (Rouen-1 47 His; Rouen-2 47 Ser) at this residue. These results confirm the view that Arg-47 is an important residue in heparin binding. No dimers of Barcelona-2 were observed suggesting that steric hindrance of the new cysteine at residue 47 prevents dimerisation.

Amino Acid Sequence↗
PubMed1988

Antithrombin Glasgow, 393 Arg to His: a P1 reactive site variant with increased heparin affinity but no thrombin inhibitory activity.

Antithrombin Glasgow is a hereditary abnormal antithrombin that has lost thrombin inhibitory activity. It was isolated from the plasma of a 41-year-old male with a history of thrombotic events. Antithrombin Glasgow was purified from plasma using heparin-Sepharose chromatography at pH 7.4 eluting with increasing concentrations of NaCl. The normal protein eluted with 0.9 mol/l NaCl and Glasgow with 1.05 mol/l NaCl. Electrophoresis in agarose at pH 8.6 showed the variant to migrate more anodally than normal. The C-terminal small fragment resulting from catalytic cleavage with elastase between P3 and P4 of the reactive loop was isolated and sequenced. This showed the replacement of the arginine at residue 3 by a histidine. This is residue 393 in the intact molecule. The findings suggest that heparin, on binding, interacts indirectly with the reactive centre region of antithrombin.

Adult↗
PubMed1988

Proposed heparin binding site in antithrombin based on arginine 47. A new variant Rouen-II, 47 Arg to Ser.

Antithrombin Rouen-II, a new inherited variant of antithrombin-III, was found in two members of a family with no definite history of thrombosis. The subjects had normal antigenic concentrations of antithrombin and normal progressive inhibitory activity. However, the variant had defective heparin and heparan sulfate cofactor activities, and was not activated by a synthetic pentasaccharide representing the minimum heparin sequence. The abnormal antithrombin was isolated using heparin-Sepharose chromatography, and on electrophoresis at pH 8.6 migrated more anodally than normal. Two-dimensional peptide mapping of tryptic and Staphylococcus aureus V8 protease digests was performed and the abnormal peptide was located by tryptophan staining. Amino acid sequence studies demonstrated a substitution of arginine at residue 47 by a serine. Evidence strongly suggests that arginine 47 is a prime heparin binding site in antithrombin and that it forms part of a proposed positively charged linear site (to which heparin binds) that stretches across the surface of the molecule from the A to the D helix.

Adult↗
PubMed1987

Heparin binding defect in a new antithrombin III variant: Rouen, 47 Arg to His.

Antithrombin III (AT-III) Rouen is a hereditary abnormal antithrombin with normal progressive inhibitory activity and reduced heparin cofactor activity. It was isolated from the plasma of a woman who suffered a sudden idiopathic sensorineural hearing loss and balance impairment. There was no familial history of thrombosis. By heparin-Sepharose chromatography, AT-III Rouen was separated from the normal antithrombin on elution with increasing concentrations of NaCl. AT-III Rouen eluted earlier than is normal at both pH 7.4 and pH 6.0. At the lower pH, the antithrombins bound more avidly to the column, with the abnormal AT-III eluting closer to the normal than at the higher pH. Two-dimensional peptide mapping of tryptic and Staphylococcus aureus V8 protease digests of carboxymethylated antithrombins was performed on thin-layer silica plates. The abnormal peptide was located by tryptophan staining, and amino acid analysis and sequence studies demonstrated a substitution of an arginine at residue 47 for a histidine. Results from this study suggest that replacement of arginine 47 by a partially positively charged histidine has less effect on the heparin binding affinity than dose replacing it with a neutral cysteine side chain as in AT-III Toyama, in which no heparin binding was observed. In addition, heparin binding per se is not a sufficient condition to activate AT-III.

Adult↗
PubMed1984

Circulating proalbumin associated with a variant proteinase inhibitor.

The unique finding of normal proalbumin in human plasma provides an insight into the mechanism of propeptide cleavage. Proalbumin, present as 1-5% of the total albumin, was found in a boy whose prime problem was the presence of a mutant proteinase inhibitor, alpha 1-antitrypsin Pittsburgh (358 Met----Arg) [2]. The inferred structure of human proalbumin was confirmed as Arg-Gly-Val-Phe-Arg-Arg-Alb. On incubation with various enzymes (trypsin, tryptase, thrombin, chymotrypsin, chymase and cathepsin B), only trypsin was capable of converting proalbumin to albumin. There was no conversion when proalbumin was incubated with whole blood, plasma or serum. However, intravenous injection of proalbumin into a rat resulted in complete conversion to albumin, the half-life of this process being 6 h. We conclude that propeptide cleavage is dependent on a serine proteinase which is inhibited intracellularly, by the mutant inhibitor, and that all the albumin in the boy was secreted as proalbumin, but was subjected to a separate cleavage process after export from the hepatocyte.

Adolescent↗
PubMed1983

Mutation of antitrypsin to antithrombin. alpha 1-antitrypsin Pittsburgh (358 Met leads to Arg), a fatal bleeding disorder.

Our previous studies predicted a functional relationship between the plasma proteins alpha 1-antitrypsin and antithrombin III. To elucidate this relationship we investigated the plasma of a 14-year-old boy who had died from an episodic bleeding disorder. A variant alpha 1-antitrypsin was identified in which the methionine at position 358 had been replaced by an arginine. This had converted the alpha 1-antitrypsin from its normal function as an inhibitor of elastase to that of an inhibitor of thrombin. This finding indicates that the reactive center of alpha 1-antitrypsin is methionine 358, which acts as a bait for elastase, just as the normal reactive center of antithrombin III is arginine 393, which acts as a bait for thrombin. The independence of the new thrombin inhibitor from heparin control explains the bleeding disorder; it also indicates that heparin normally acts directly on antithrombin III, revealing its inherent inhibitory activity. The episodic nature of the bleeding was a consequence of the mutant protein's being an acute-phase reactant, the level of which increased several-fold after trauma.

Adolescent↗
PubMed1982

Structure and variation of human alpha 1-antitrypsin.

The sequence of alpha 1-antitrypsin is in keeping with its role as a tissue scavenger of leukocyte elastase. Two abnormal variants commonly present in Europeans cause a deficiency that predisposes them to a progressive loss of lung elasticity. The nature of the reactive centre helps explain why cigarette smoking greatly accelerates the onset and severity of this degenerative process to give the disease emphysema.

Amino Acid Sequence↗