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P O'Toole

Publications and source records attributed to P O'Toole.

9 recordsLinked to original sources
PubMed1998

Detection of dimers of dimers of human leukocyte antigen (HLA)-DR on the surface of living cells by single-particle fluorescence imaging.

The technique of single-particle fluorescence imaging was used to investigate the oligomeric state of MHC class II molecules on the surface of living cells. Cells transfected with human leukocyte antigen (HLA)-DR A and B genes were labeled at saturation with a univalent probe consisting of Fab coupled to R-phycoerythrin. Analysis of the intensities of fluorescent spots on the cell surface revealed the presence of single and double particles consistent with the simultaneous presence of HLA-DR heterodimers and dimers of dimers. The proportion of double particles was lower at 37 degrees C than at 22 degrees C, suggesting that the heterodimers and dimers of dimers exist in a temperature-dependent equilibrium. These results are discussed in the context of a possible role for HLA-DR dimers of dimers in T cell receptor-MHC interactions. The technique is validated by demonstrating that fluorescence imaging can distinguish between dimers and tetramers of human erythrocyte spectrin deposited from solution onto a solid substrate. The methodology will have broad applicability to investigation of the oligomeric state of immunological and other membrane-bound receptors in living cells.

Cell Line↗
PubMed1997

Enhancement of photodynamic therapy in gastric cancer cells by removal of iron.

BACKGROUND: Aminolaevulinic acid (ALA) is an endogenous substrate in the haem biosynthetic pathway. Protoporphyrin IX (PPIX), the immediate haem precursor in the pathway, has photoexcitable properties. Exogenous ALA has been used previously as a precursor agent in photodynamic therapy (PDT). Its main advantage is a short half-life and hence reduced incidence of skin photosensitivity. ALA can be toxic, however, causing, for example, transient increases in liver enzyme concentrations when given systemically and this may be dose related. AIM: To assess whether accumulation of PPLX and ultimately the efficacy of PDT could be improved by modulating both ends of the haem biosynthetic pathway. METHODS: Gastric cancer cells (MKN 28) were incubated with ALA (0-1000 mumolar) and desferrioxamine (0-800 mumolar) for 24 hours before exposure to argon-pumped dye laser (630 nm) at different energy levels (0-40 J/cm2). Cell viability was assessed by use of the methyl-tetrazolium (MTT) assay four hours after exposure to light. RESULTS: Total PPIX accumulation increased linearly with increasing extracellular concentrations of ALA up to 1 mmolar (r = 0.973, p < 0.005). Adding 200 molar of desferrioxamine trebled PPIX accumulation over the same period of incubation. Cell viability after exposure to light decreased with low doses (0-30 mumolar) of desferrioxamine (r = 0.976, p = 0.024). However, higher doses of desferrioxamine (more than 40 molar) seemed to confer a protective effect against PDT. CONCLUSION: PDT using ALA can be improved by removal of available iron with desferrioxamine. The reason for the protective effect of desferrioxamine seen at higher doses is not clear.

Aminolevulinic Acid↗
PubMed1992

Two major classes in the M protein family in group A streptococci.

The M protein family of molecules in the group A streptococcus comprises a number of cell surface proteins that interact with the immune system of the host. One of the proteins in this family is the IgA receptor Arp4, which has C repeats similar to those that characterize the known M proteins. The streptococcal strain expressing Arp4 also expresses a second immunoglobulin-binding protein, Mrp4, which is shown here to be encoded by a gene located immediately upstream of the gene for Arp4. In addition to binding IgG, Mrp4 also binds fibrinogen, a property ascribed to M proteins. DNA sequence analysis demonstrated that the Mrp4 protein indeed is a member of the M protein family, but it was unexpectedly found to have a type of repeat that is identical to the A repeat described for FcRA76, a partially sequenced streptococcal Fc receptor. Purified FcRA76 was shown to bind fibrinogen and IgG, like Mrp4. These data show that the known molecules in the M protein family can be divided into two classes, A and C, according to the type of repeat region found. Hybridization studies with a panel of clinical isolates indicate that many streptococcal strains express class A and class C proteins, whereas some strains express only class C proteins. Class A molecules show amino-terminal sequence variation, like class C molecules, which suggests that proteins of both classes are targets for the immune response.

Amino Acid Sequence↗
PubMed1992

Many group A streptococcal strains express two different immunoglobulin-binding proteins, encoded by closely linked genes: characterization of the proteins expressed by four strains of different M-type.

Most group A streptococcal strains are able to bind immunoglobulin (Ig) in a non-immune manner, and the majority of these strains bind both IgA and IgG. Using molecular cloning and immunochemical techniques, we have purified and characterized the Ig Fc-receptors expressed by four such strains. Two of the strains express a novel type of receptor, designated protein Sir, which binds IgA and IgG of all subclasses, and therefore has broader reactivity than any Fc-receptor previously described. The other two strains express protein Arp, a receptor that binds IgA of both subclasses, and also binds polyclonal IgG weakly. Characterization of the weak IgG-binding ability of protein Arp shows that it binds only some monoclonal IgG proteins, in particular those of the IgG3 subclass. The four strains studied here were unexpectedly found to also express a second Ig-receptor, called protein Mrp, encoded by a gene closely linked to the gene for the first receptor. The Mrp protein does not bind IgA, but it binds IgG molecules of the IgG1, IgG2 and IgG4 subclasses, and it also binds fibrinogen. Binding of fibrinogen has been reported to be a characteristic property of streptococcal M proteins, which suggests that the Mrp protein may be an M protein that also binds Ig. Taken together, all available evidence now indicates that most strains of group A streptococci express two different Ig-binding proteins, encoded by closely linked genes.

Amino Acid Sequence↗
PubMed1991

High-affinity binding of the basement membrane proteins collagen type IV and laminin to the gastric pathogen Helicobacter pylori.

The ability of 16 isolates of the human gastroduodenal pathogen Helicobacter pylori to bind 125I-radiolabelled tissue proteins was quantitated by liquid-phase assay. While capable of binding generally low levels of collagen types I and II, vitronectin, and fibronectin (average binding, 8%; highest binding, 23%), the various H. pylori isolates were good binders of the basement membrane proteins collagen type IV and laminin (average binding, 27%; highest binding, 60%). Campylobacter species tested bound lower levels of collagen type IV and laminin (average binding, 12%; highest binding, 17%). Trypsin and proteinase K treatment of H. pylori cells markedly reduced the binding of collagen type IV and laminin, as did heat treatment, suggesting that the binding of basement membrane proteins is mediated by bacterial surface proteins. Binding of both basement membrane proteins was rapid and saturable. 125I-collagen type IV binding to H. pylori 915 was inhibited by preincubation with unlabelled collagen type IV but was not inhibited by laminin or a number of other proteins. Once bound, radiolabelled collagen type IV but was not displaced by an excess of unlabelled collagen type IV, indicating that the binding interaction was of high affinity. Binding of laminin was partially reversible, and analysis in a solid-phase nonradiolabel assay showed that the interaction was of high affinity, with a Kd of 7.9 nM. This interaction was affected by salt, indicating the presence of a hydrophobic component in the ability of H. pylori to bind laminin.

Binding Sites↗
PubMed1989

Binding to collagen by Yersinia enterocolitica and Yersinia pseudotuberculosis: evidence for yopA-mediated and chromosomally encoded mechanisms.

Binding of Yersinia enterocolitica and Yersinia pseudotuberculosis strains to type I, II, and IV collagens has been studied. Wild-type strains which harbored the 40- to 50-megadalton virulence plasmid specifically bound all three types of collagen. Curing of the virulence plasmid or Tn5 insertion in the yopA gene encoding the temperature-inducible outer membrane protein YOP1 abolished the binding of all three collagen types to Y. enterocolitica and type I and II collagens to Y. pseudotuberculosis. Full binding capacity was restored by introduction of the yopA gene into nonbinding Yersinia strains. Binding of type I, II, and IV collagens was expressed in Escherichia coli constructs harboring the yopA gene of either Y. enterocolitica or Y. pseudotuberculosis. The interaction of bacterial cells with type I collagen could be blocked by nonradiolabeled native collagens or denatured collagen but not with other serum and connective-tissue proteins. Unlabeled collagen could not displace bound radiolabeled collagen. The binding was inhibited by YOP1-specific polyclonal antibodies, in contrast to normal rabbit serum. The interaction was rapid and was quite resistant to heat treatment, to proteolytic enzymes, to various pHs in both acidic and alkaline ranges, and to the chaotropic agent urea. We propose that this newly identified interaction may be involved both in the first steps of the pathogenesis and in the complications of Yersinia infections affecting connective tissue.

Bacterial Outer Membrane Proteins↗