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Biomedical subjects

I D Walker

Publications and source records attributed to I D Walker.

At least 109 records · Page 6Linked to original sources
PubMed1993

Molecular cloning of a member of the gene family that encodes pMGA, a hemagglutinin of Mycoplasma gallisepticum.

A hemagglutinin with an M(r) of 67,000 (pMGA) from Mycoplasma gallisepticum S6 was purified by using monoclonal antibody affinity chromatography. Purified pMGA was treated with a number of enzymes, the resultant peptides were purified, and their amino acid sequence was determined by using an Applied Biosystems (model 471A) protein sequencer. The DNA sequence encoding two peptides was used to dictate the sequences of synthetic oligonucleotides which were used to screen a library of EcoRI-cut M. gallisepticum DNA in pUC18. A clone reactive to both probes was isolated and found to contain a recombinant insert of 10 kb. The clone was mapped by using restriction endonucleases and fragments subcloned into pUC18 for DNA sequencing. Analysis of part of the DNA sequence revealed an open reading frame containing 1,941 nucleotides which encoded 647 amino acids. The amino terminus was preceded by a putative leader sequence of 25 amino acids. A promoter region preceding the putative start codon GUG was also located. This gene would encode a mature protein of 67,660 Da. There were a number of differences between the predicted amino acid sequence and that determined by direct peptide sequencing. Also, two tryptic peptides of pMGA were not found in the DNA sequence. This suggested that the cloned gene did not encode pMGA but did encode a homolog (pMGA1.2). Furthermore, downstream of pMGA1.2 was a region of DNA encoding a leader sequence followed by an amino acid sequence with high homology to that encoded by the pMGA1.2 gene. The presence within M. gallisepticum of a family of pMGA genes is inferred from the DNA sequence and Southern transfer data. A possible role for this gene family in immune evasion is discussed.

Amino Acid Sequence↗
PubMed1992

Plasminogen levels in healthy volunteers--influence of age, sex, smoking and oral contraceptives.

There is considerable doubt as to the importance of reduced plasminogen (PLG) activity as a risk factor for venous thrombosis. In the present study we have identified a wide range of PLG activities (25-200%) in a cohort of 9,611 blood donors. Males and females not taking hormonal contraceptives show a similar distribution of PLG, however, variation related to age appears to follow a different pattern in males and females. These differences are of doubtful clinical importance as are differences related to smoking. In contrast, females taking hormonal contraceptives or hormone replacement therapy (HRT) have up to 25% higher mean PLG levels in younger females but a less marked elevation (10%) is seen in 40-50 year olds. A PLG activity < 65% was recorded in 61 donors, none of whom appeared to have a history of thrombosis. These findings do not support the notion that reduced PLG is an important thrombophilic risk factor, however, further investigation of the donors with low PLG is required.

Adolescent↗
PubMed1992

SP-40,40, a protein involved in the control of the complement pathway, possesses a unique array of disulphide bridges.

SP-40,40 is a two-chain serum protein which acts in vitro as a potent inhibitor of the assembly of the membrane attack complex of human complement. It contains 10 cysteine residues, the numbers and locations of which are conserved in several mammalian species. Evidence is presented that all the cysteine residues are involved in interchain (alpha-beta) disulphide bonds. There are no free cysteine residues. The disulphide bond motif established in this study for SP-40,40 is unique and bears no obvious homology to those complement components whose disulphide bonds have been assigned, nor is there any homology apparent between SP-40,40 and other multi-chain proteins containing disulphide bonds.

Amino Acid Sequence↗
PubMed1992

Characterization of a major hemagglutinin protein from Mycoplasma gallisepticum.

Mycoplasma gallisepticum cell membranes were used to immunize mice to produce monoclonal antibodies to cell surface proteins. Three monoclonal antibodies were chosen for further characterization. All three reacted in immunoblots with an M. gallisepticum protein band of M(r) approximately 67,000 (designated pMGA). By using immunoelectron microscopy, pMGA was shown to be located on the cell surface. When M. gallisepticum whole cells were treated with up to 250 micrograms of trypsin per ml for 30 min, the only major protein lost from the cell surface as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblot transfer was pMGA. Two of the pMGA-specific monoclonal antibodies inhibited hemagglutination of chicken erythrocytes by M. gallisepticum S6, suggesting a role for pMGA in the attachment of M. gallisepticum to chicken erythrocytes. Sequencing the amino terminus of pMGA yielded 17 amino acids with no significant homology with the Mycoplasma pneumoniae attachment protein P1 or any other protein in the GenBank, Swiss-Prot, and EMBL data bases.

Amino Acid Sequence↗
PubMed1991

The apolipoprotein A-I binding protein of placenta and the SP-40,40 protein of human blood are different proteins which both bind to apolipoprotein A-I.

A complement-associated protein SP-40,40, which is a normal constituent of human blood, binds to the main apoprotein, apoA-I, of high density lipoprotein (HDL). This protein, which is identical to apolipoprotein J, was compared to another apoA-I binding protein purified from human placenta. Immunologically the two apoA-I binding proteins are different.

Amino Acid Sequence↗
PubMed1991

Antithrombin Cambridge II, 384 Ala to Ser. Further evidence of the role of the reactive centre loop in the inhibitory function of the serpins.

Four unrelated individuals have been identified with an identical antithrombin variant, associated in one of them with episodes of recurrent venous thromboses. In each case, the plasma antithrombin concentration was normal and the only function abnormality was a minor but consistent decrease in the heparin-induced thrombin inhibition suggesting a mutation at or near the reactive centre of the molecule. Amplification and direct sequencing of exon 6 showed a G----T mutation at nucleotide 1246, which corresponds to a substitution of a serine for an alanine at residue 384. This is one of a series of conserved alanines that form the stalk to the reactive centre loop. The observed changes in this variant are compatible with recent structural studies that infer that mobility of this stalk with partial re-entry into the A-sheet of the molecule is necessary for optimal inhibitory activity.

Aged↗
PubMed1991

Management of thrombophilia in pregnancy.

Thromboembolism remains a major cause of maternal morbidity and mortality. The incidence of thrombosis associated with pregnancy is stated to be around 0.09%1 but is greater in women with familial or acquired thrombophilia. Around 50% of pregnancies in women with antithrombin III deficiency are complicated by thrombosis. Anticoagulation throughout pregnancy and the puerperium is recommended in women with antithrombin III deficiency. Because thrombosis is less common in women with protein C or protein S deficiency less aggressive management may be appropriate during pregnancy but anticoagulation post partum is generally recommended. The most important acquired thrombophilic abnormality is the development of antiphospholipid antibodies ('lupus anticoagulants'). Women with these antibodies may present major problems but no clear guidelines for their management currently exist. The majority of women with a history of thrombosis have no identifiable haemostatic abnormality. Management of pregnancy in these patients depends on individual circumstances.

Blood Coagulation Disorders↗
PubMed1991

Antithrombin Glasgow II: alanine 382 to threonine mutation in the serpin P12 position, resulting in a substrate reaction with thrombin.

A female with recurrent thrombosis was found to have a functional abnormality of antithrombin, with a ratio of functional to immunological activity in plasma of approximately 50%. Crossed immunoelectrophoresis in the presence of heparin was normal, indicating an abnormality of the reactive site, rather than the heparin binding domain. Accordingly, the antithrombin was isolated by heparin-Sepharose chromatography: this produced a mixture of normal and variant antithrombin, as the patient was heterozygous for the abnormality. To remove the normal component, the antithrombin was passed through a column of thrombin-Sepharose. On sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), prior to its application to thrombin-Sepharose, the antithrombin migrated as a single band with identical mobility to that of normal antithrombin. After thrombin-Sepharose, the purified variant component was proteolysed, and migrated as two components, one with a reduced and one with enhanced mobility under non-reducing conditions. This demonstrated that the variant was unable to form stable inhibitor-thrombin complexes and was cleaved in a substrate reaction with thrombin. One site of cleavage was unambiguously ascertained to be the Arg 393-Ser 394 reactive site bond, by NH2 terminal sequencing of the cleaved variant antithrombin: 10 steps beginning at the P1' position, Ser-Leu-Asn-Pro-Asn-Arg,..., were clearly identified. The mutation responsible for this defect was studied by polymerase chain reaction (PCR) amplification of exon 6 of the antithrombin gene and direct sequencing of the amplified product. The presence of both a G and A in the first position of codon 382, identified the mutation GCA to ACA, which results in the substitution of Ala 382 to Thr. This is identical to that reported for antithrombin Hamilton (Devraj-Kizuk et al, 1988), although antithrombin gene polymorphism analysis suggests that the antithrombin Glasgow II mutation has arisen independently. We have recently shown (Caso et al, 1991) that mutation at a nearby position, Ala 384 to Pro, also transforms another variant, antithrombin Vicenza/Charleville, into a substrate for thrombin. The present results with antithrombin Glasgow II suggest that all the alanine residues at the base of the reactive site loop in positions P12-10 may be important for the formation of a stabilized inhibitor-thrombin complex.

Adult↗
PubMed1990

Human seminal clusterin (SP-40,40). Isolation and characterization.

Molecular cloning of the human complement inhibitor SP-40,40, has revealed strong homology to a major rat and ram Sertoli cell product, sulfated glycoprotein-2, known also as clusterin. This study reports the purification and characterization of human seminal clusterin. Two-dimensional gel electrophoresis revealed charge differences between clusterin purified from semen and the serum-derived material. Both preparations demonstrate comparable hemagglutination (clustering) activity and inhibition of C5b-6 initiated hemolysis. The average clusterin concentration in normal seminal plasma is considerably higher than that found in serum. Mean seminal plasma clusterin concentrations were significantly lower in azoospermia caused by obstruction or seminiferous tubule failure than with oligospermia or normospermia. Only men with vasal agenesis had undetectable seminal clusterin, suggesting that some of the seminal clusterin is produced by the seminal vesicles. Immunofluorescence of human spermatozoa revealed that clusterin was detected on 10% of spermatozoa, predominantly those that were immature or had abnormal morphology. A pilot study of 25 patients suggests that seminal clusterin concentration, together with sperm motility and morphology, is correlated with the fertilization rate in vitro. The function of seminal clusterin is unknown. Its extensive distribution in the male genital tract and its high concentration in seminal plasma suggests an important role in male fertility.

Animals↗
PubMed1990

A Scottish Hebridean antithrombin III deficient family--twelve years on.

The members of this family with ATIII deficiency have been followed for at least 12 years (1976-1989). All those with previous venous thrombo-embolism have been free from recurrence when on warfarin. During the half century 1931-1981, all 11 full term pregnancies in four affected patients were associated with venous thromboembolism; one patient was enigmatic having one full term pregnancy, without thrombotic event; between 1982 and 1989 three pregnancies have been actively managed with no clinical thrombosis. Management involved use of monitored, self administered, subcutaneous heparin before or very soon after conception and throughout pregnancy (warfarin having been stopped), planned delivery under cover of intravenous antithrombin III, reduction of heparin dosage at delivery and reintroduction of warfarin in the puerperium. The recognised hazards of heparin therapy in pregnancy did not occur. The involvement of the arterial system is reviewed. Clinical evidence provides tentative suggestions on (a) possible additional risk of cigarette smoking (b) avoidance of venography (c) avoidance of varicose vein surgery. A probe is now available for the defective antithrombin III gene in this family, but there has been no occasion yet to apply this in antenatal diagnosis.

Abortion, Spontaneous↗
PubMed1989

The alpha-chain of murine CD8 lacks an invariant Ig-like disulfide bond but contains a unique intrachain loop instead.

The CD8 Ag is a cell surface heterodimer which demarcates predominantly cytotoxic T cells which are restricted by class I MHC Ag. The disulfide bonds within the murine structure were assigned in this study and the alpha-beta-interchain bond involves one or more cysteine residues located in each chain proximal to the plasma membrane or included within it. The location of the intrachain disulfide loop within the CD8 beta-chain confirms its proposed structural homology to an IgV domain but no corresponding disulfide loop is present within the alpha-chain. The invariant IgV disulfide loop has been replaced by a unique, short loop involving an unusual cysteine which is conserved in the CD8 alpha-chains of man, mouse, and rat. Despite its lack of precedent in other Ig-related structures, this unusual disulfide loop can be parsimoniously accommodated into a modified domain which has retained the major features of the Ig structural motif.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and characterization of the novel, human complement-associated protein, SP-40,40: a link between the complement and reproductive systems.

The cDNA sequence encoding the human complement-associated protein, SP-40,40, is reported. The two chains of SP-40,40 are coded in a single open reading frame on the same mRNA molecule, indicating the existence of a biosynthetic precursor protein which matures post-synthetically by the proteolysis of at least one peptide bond. The precursor is preceded by a signal sequence for vectorial export and contains six N-linked glycosylation sites distributed equally between the two chains of the structure. The sequence of the SP-40,40 precursor bears a 77% identity to a rat sulphated glycoprotein-2 (SGP-2) which is the major secreted product of Sertoli cells. The presence of SP-40,40 within human seminal plasma at levels comparable to those in serum was demonstrated, indicating that SP-40,40 and SGP-2 are serum and seminal forms of the same protein. A sequence of 23 amino acids within the beta-chain of SP-40,40 exhibited significant homology to corresponding segments located within complement components C7, C8 and C9. The short cysteine-containing motif represented the only evidence of a possible vestigial relationship between SP-40,40 and other complement components. The precise role of SP-40,40 is not known in either blood or semen but the present findings document an intriguing link between the immune and the reproductive systems.

Amino Acid Sequence↗