Biomedical subjects

J F Wright

Publications and source records attributed to J F Wright.

At least 37 records · Page 2Linked to original sources

PubMed1994

Prothrombinase assembly on an enveloped virus: evidence that the cytomegalovirus surface contains procoagulant phospholipid.

In contrast to quiescent cells, we currently report that purified cytomegalovirus (CMV), strain AD169, constitutively expresses phosphatidylserine (PS)-like procoagulant activity. Initial evidence for this came from the observation of a CMV-dependent decrease in factor Xa clotting times. In a purified system, the assembly of a functional complex between factor Xa and the cofactor Va to form prothrombinase was found to be dependent on the addition of CMV. The corresponding dense bodies (DB) and noninfectious enveloped particles had similar activity. Quantification of the total virion and DB phospholipid, and comparison of prothrombin conversion rates to experiments conducted using known concentrations of PS-containing vesicles showed that 8.5% and 7.2% of the CMV and DB phospholipid, respectively is procoagulant. Direct binding studies of 125I-labeled factor Xa, active site-blocked factor Xa, or the zymogen factor X, with virions or DB showed a single class of Ca(2+)-dependent sites with dissociation constants in the order of 10(-7) MOL/l. Immunogold electron microscopy confirmed the specificity of the CMV/factor Xa association. Cumulatively, these data suggest that the CMV surface contains the necessary procoagulant phospholipid for coagulation enzyme complex assembly. This may enable CMV (and possibly other enveloped viruses) to bypass an important physiologic regulatory mechanism for the production of thrombin.

Animals↗

PubMed1994

C1q binding properties of monomer and polymer forms of mouse IgM mu-chain variants. Pro544Gly and Pro434Ala.

The effect of replacing proline with alanine at position 434 in the C mu 3 domain (P434A) and with glycine at position 544 in the C mu 4 domain (P544G) of the mu-chain of mouse IgM has been studied. The P434A substitution results in the loss of measurable complement-mediated cytolytic activity (CML) and a decrease in the association rate constant at low ionic strength (mu = 0.06), that results in a diminished Ka for C1q binding to P434A IgM bound to haptenated cells (0.4 x 10(9) M-1). Binding of C1(qr2s2) could not be detected. In contrast, replacement of proline at 544 had no measurable effect on the cytolytic or C1q/C1 binding properties of the polymeric molecule, supporting the view that the C mu 3 domain is important in C1q binding and CML. The secreted monomeric subunit of P544G was not able to mediate CML. Also, whereas hapten-bound P544G polymer bound C1q with a functional affinity of 1.5 x 10(9) M-1 at low ionic strength (mu = 0.06), similar to that observed with wild-type polymer (1.7 x 10(9) M-1) and wild-type IgG monomer (4.7 x 10(9) M-1), no C1q binding was detected with the P544G IgM monomer. This could not be attributed to differences in glycosylation. Inasmuch as the P544G mutation per se had no effect on the C1q binding properties of the polymer, we conclude that unlike IgG, aggregation does not sufficiently enhance the avidity of IgM monomer to enable it to activate complement. Augmentation of the site must occur during polymerization or when the IgM binds to Ag.

Animals↗

PubMed1994

An endothelial cell-surface form of annexin II binds human cytomegalovirus.

Human cytomegalovirus was shown to bind to human umbilical vein endothelial cells in a specific, saturable and calcium-dependent manner (Kd = 7.9 pM (4 degrees C), 6469 virus binding sites/cell). Affinity adsorption of detergent-prepared lysates of surface-radiolabeled endothelial cells to virions resulted in the identification of cell-derived proteins of approximate M(r) 36,000 and 32,000 that bound cytomegalovirus. Protein sequencing of peptides obtained by cyanogen bromide cleavage demonstrated that the 36 kDa protein corresponded to human annexin II, and the 32 kDa protein was likely a degradation product. Purified annexin II was demonstrated to bind directly to virions (Kd = 57 nM, 688 annexin II binding sites/virion). These results provide evidence that an endothelial cell-surface form of annexin II acts as a receptor for cytomegalovirus, and indicate a previously undescribed role for annexin II.

Amino Acid Sequence↗

PubMed1992

Identification and partial characterization of a low affinity metal-binding site in the light chain of tetanus toxin.

Tetanus toxin was shown to contain a metal-binding site for zinc and copper. Equilibrium dialysis binding experiments using 65Zn indicated an association constant of 9-15 microM, with one zinc-binding site/toxin molecule. The zinc-binding site was localized to the toxin light chain as determined by binding of 65Zn to the light chain but not to the heavy chain after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to Immobilon membranes. Copper was an efficient inhibitor of 65Zn binding to tetanus toxin and caused two peptide bond cleavages in the toxin light chain in the presence of ascorbate. These metal-catalyzed oxidative cleavages were inhibited by the presence of zinc. Partial characterization of metal-catalyzed oxidative modifications of a peptide based on a putative metal-binding site (HELIH) in the toxin light chain was used to map the metal-binding site in the protein.

Amino Acid Sequence↗

PubMed1991

Proteolytic fragmentation of tetanus toxin by subcellular fractions of JY, a B lymphoblastoid cell line.

Proteolysis of 125I-labelled tetanus toxin by subcellular fractions from an Epstein-Barr-virus-transformed B lymphoblastoid cell line, JY, was investigated. Fractions enriched in lysosomes and plasma membranes cleaved the toxin molecule at several sites, with a pH optimum of 5.5. N-Terminal sequence analysis of Mr-81,000, -45,000 and -35,000 proteolytic fragments indicated cleavage of the Asp-460-Leu-461, Asp-872-Glu-873 and Ile-1013-Thr-1014 peptide bonds, all sites located within the heavy chain of the toxin molecule. Additional sites near the C-terminus of the heavy chain, giving rise to low-Mr peptides, were implicated. The toxin light chain was more resistant to proteolysis. A similar pattern of fragmentation was observed with tetanus toxin biosynthetically radiolabelled with 14C-labelled amino acids, showing that the proteolysis was not an artifact caused by iodination. The proteolytic activity was inhibited by the serine proteinase inhibitor di-isopropyl phosphorofluoridate, thiol-blocking proteinase inhibitors N-ethylmaleimide and iodoacetamide, and by EDTA. These results represent a preliminary characterization of the processing in vitro of tetanus toxin by an antigen-presenting cell line.

B-Lymphocytes↗

PubMed1991

Formation of covalent C3b-tetanus toxin complexes: a tool for the in vitro study of antigen presentation.

A novel method is described for the formation and purification of covalent complexes between the complement component C3b and an antigen (tetanus toxin, TT), using purified proteins in fluid phase. C3b is generated in situ by tryptic cleavage of C3 after co-precipitation of C3 and TT in the presence of polyethylene glycol. Various parameters were analysed to optimize complex formation; under conditions which minimized the formation of covalent C3b multimers, 30% and 8% respectively of C3b and TT were incorporated into covalent one-to-one complexes which were purified using gel filtration chromatography. The linkage was localized between the alpha' chain of C3b and either the H or L chain of TT; it required the in situ formation of C3b and was partially destroyed by 1 M hydroxylamine. Spontaneous dissociation of the complex could be partly avoided by HgCl2, a thiol reagent which inhibits the esterase-like activity of bound C3b. These findings suggest the involvement of the reactive carbonyl of nascent C3b with hydroxyl groups of TT. Such C3b-TT complexes provide a defined tool to analyse the influence of antigen-bound C3b on antigen addressing and intracellular processing by antigen-presenting cells.

Animals↗

PubMed1990

C1 binding by mouse IgM. The effect of abnormal glycosylation at position 402 resulting from a serine to asparagine exchange at residue 406 of the mu-chain.

We have previously shown that IgM-Asn406, a mutant IgM which has asparagine in place of the serine which is normally found at position 406, also has an abnormally glycosylated mu-chain and is defective in complement-dependent cytolysis. Here we show by analyzing cyanogen bromide fragments from normal and mutant mu-chains that the site of abnormal glycosylation is at the neighboring position, Asn402. The cytolytic defect was shown to be due to impaired C1 binding. At physiological ionic strength, the C1 binding defect was estimated to be 12-fold, which correlates well with the measured defect in cytolytic activity; also, the severity of the defect in C1 binding by the mutant protein decreases with decreasing ionic strength. Kinetic studies showed that the difference in affinities is due to a proportional difference in the association rate for C1q. By comparing IgM made in the presence and absence of deoxymannojirimycin, we show further that the defect in cytolytic activity derives mostly from the abnormal oligosaccharide.

Amino Acid Sequence↗

PubMed1990

Some unusual features of mycobacteriosis in the cichlid fish Oreochromis mossambicus.

Adult, aquarium-reared Oreochromis mossambicus (Tilapia mossambica), which were naturally infected with Mycobacterium marinum, displayed non-healing skin ulcers and other clinical signs considered to be typical of piscine mycobacteriosis. However, in addition, they frequently had melanotic foci in the skin and spleen, due to the presence of pigment cells surrounding the cutaneous and splenic inflammation. Such melanotic foci have never been reported in response to mycobacteriosis. All fish also have variable numbers of melanomacrophages which appeared to replace pancreatic acini. The relationship of the pancreatic melanomacrophages to the pathogenesis of mycobacteriosis is uncertain, but such lesions may have contributed to the chronic cachexia associated with this case.

Animals↗

PubMed1989

Chronic inflammatory cells with epithelial cell characteristics in teleost fishes.

Certain cells that participate in the chronic inflammatory response of teleost fishes have many features typical of epithelioid cells of mammals. Such features include high metabolic activity, frequent phagolysosomes, and cytoplasmic interdigitations between adjacent cells; however, the epithelioid granulomas formed in response to certain diseases in teleost fishes also have several features associated with epithelial cells. Cases of ulcerative mycosis or acid-fast bacterial infection in Atlantic menhaden (Brevoortia tyrannus), fungal infection in silver perch (Bairdiella chrysoura), and mycobacteriosis in Mozambique tilapia (Oreochromis mossambicus) had epithelioid cells that were joined together by well-formed desmosomes with tonofilaments. "Mature granulomas" of the ulcerative mycosis-infected menhaden stained positively for cytokeratin, a cytoskeletal protein that is considered to be highly specific for epithelial cells. The consistent presence of these heretofore unrecognized epithelial features suggest that they may be characteristic of certain types of cells participating in piscine chronic inflammation.

Animals↗

PubMed1988

A mitochondrial intergenic mutation affecting processing of specific yeast mitochondrial transcripts.

The mutation in the temperature-conditional mit- mutant h56, mapped previously to the var1 gene region of Saccharomyces cerevisiae mitochondrial DNA, results in a specific inhibition of var1 protein synthesis in cells incubated at the non-permissive temperature, 36 degrees C (1). We have now characterized the mutation present in mutant h56 by DNA sequencing and found it to be an A to T transversion located 109 nucleotides upstream of the var1 reading frame. Two spontaneous revertants of mutant h56 restore the parental strain sequence at residue -109, confirming that this single base change within the 5'-untranslated region of the var1 mRNA is responsible for defective synthesis of the var1 protein. A comparison of var1 transcripts in the parental and mutant strains has shown that the mutation specifically blocks formation of var1 mRNA at 36 degrees C and leads to accumulation of precursor transcripts. Expression of the oli1 gene, co-transcribed with the var1 gene in primary transcripts, is not affected. It is concluded that the mutation in mutant h56 alters the secondary structure of the precursor RNA, inhibiting an endonucleolytic cleavage required to generate the 5' end of var1 mRNA.

Base Sequence↗

PubMed1988

C1 binding by murine IgM. The effect of a Pro-to-Ser exchange at residue 436 of the mu-chain.

We have examined a defect in complement activation in a mutant trinitrophenyl-binding pentameric murine monoclonal IgM which has serine replacing the proline normally found at position 436 in the protein. The mutant protein showed equivalent hapten binding but a 100-fold decreased ability to initiate complement-dependent lysis of trinitrophenyl-coupled erythrocytes at physiological ionic strength (mu = 0.15). C4b deposition mediated by the mutant protein was impaired to a similar degree. C1 bound by the mutant protein showed C1s to C1-s conversion, suggesting normal activation. When measured at reduced ionic strength (mu = 0.06), the C1 and C1q binding affinity of the mutant protein was approximately one-half that of the wild type. However, the C1 binding affinity of the mutant protein showed a greater dependence upon ionic strength such that at physiological ionic strength we estimate a 50-fold lower C1 binding affinity for the mutant molecule. Kinetic studies suggested that this difference in affinity was largely attributable to differences in association rates. In addition, a fixed proportion of the mutant molecules showed no C1 binding. We conclude that the defect in complement activation occurs at the level of C1 binding. Our data support a role for the C mu 3 domain (residues 340-440) in C1 binding by IgM.

Complement Activating Enzymes↗

PubMed1988

Fibrous dysplasia of the spine.

Fibrous dysplasia affecting the spine is unusual. A further 11 cases are reported and the radiological features are described. The complications and difficulties with the diagnosis of this condition are discussed.

Fibrous Dysplasia of Bone↗

PubMed1988

Spinal cord compression caused by dual pathology: a close shave with Ockham's razor.

A case of spinal cord compression in an oncology patient is presented. The compression was caused by minimal expansion of a vertebral body involved by a metastatic deposit impinging on a previously asymptomatic lipomatous spinal cord tumour. Nuclear magnetic resonance imaging clearly demonstrated both the vertebral metastasis and the intramedullary and extramedullary components of the lipomatous tumour in a single noninvasive investigation.

Aged↗

PubMed1988