The Pirbright Institute publication directory contains details of selected publications written by our researchers.

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Passos L M F, Bell-Sakyi L, Brown C G D (1998)

Immunochemical characterization of in vitro culture-derived antigens of Babesia bovis and Babesia bigemina

Veterinary Parasitology 76 (4), 239-249


Cross-reactivity between Babesia bovis and B. bigemina becomes a problem in discrimination of the two infections in endemic areas where the two species usually occur in association. With the aim of identifying candidate proteins for use as specific diagnostic tools, culture-derived components of three geographically different stocks of B. bovis (Lismore, Kwanyanga and Mexico) and one of B. bigemina (Mexico) were analyzed by immunoprecipitation using acrylamide gel electrophoresis. The approach taken was based on the analysis of S-35-methionine-labelled parasite antigens released into culture supernatant. A variety of serum samples were tested, including a panel of calf sera experimentally produced against the different stocks of Babesia, serum samples from cattle naturally infected in the field in Brazil, and a panel of anti-B. bovis monoclonal antibodies, previously characterized by the indirect fluorescent antibody test, ELISA and Western immune-blotting. Approximately 28 and 23 bands (with molecular weights ranging from 200 to 14 kDa) were detected in total protein profiles of B. bovis and B. bigemina culture supernatants, respectively, whereas no bands were seen in the uninfected red blood cell culture supernatant (negative control). The immunoprecipitation analysis showed antigenic diversity amongst the stocks of B. bovis and resulted in identification of at least five B. bovis specific antigens common to the three stocks (molecular weights of 80, 72, 58, 38 and 24 kDa) and four B. bigemina specific antigens (molecular weights of 240, 112, 50 and 29 kDa).
Preston P M, Jackson L A, Sutherland I A, Bell-Sakyi L, Wilkie G, Brown D J, Schofield J, Melrose T R, Sanderson A, Brown C G D (1998)

Theileria annulata: The expression of two novel macroshizont antigens on the surface of infected mononuclear cells differs during in vitro attenuation of a virulent cell line

Experimental Parasitology 89 (2), 228-240


The first part of this study of the biology Cal mechanisms underlying attenuation of virulent Theileria annulata macroschizont-infected cell Lines screened four pairs of T. annulata (Hisar) in vivo- and in vitro-derived macroschizont-infected cell Lines (lines) and identified a single in vivo-derived line, which induced lethal tropical theileriosis. The other seven lines were relatively avirulent. Analysis of the clinical, hematological, and parasitological responses of cattle immunized with different passages of the virulent line after in vitro culture showed that it was partly attenuated by passage (p) 50 and avirulent by p130. Clones representing the three glucose phosphate isomerase (GPI) isotypes, which constituted the newly isolated virulent culture, were obtained from p3 by limiting dilution: p50 and p130 consisted of one isotype. The second part of the study raised monoclonal antibodies (MAbs) against macroschizont-infected cells, as reagents for detecting antigenic differences between virulent and avirulent parasites, and identified two MAbs that recognized the surface of infected cells as well as macroschizonts, MAb EU1 recognized an antigen expressed by all the lines tested, whether in vitro- or in vivo-derived, whether uncloned or cloned, and irrespective of extent of subpassage in culture. MAb EU106 recognized an antigen whose expression by the virulent line and its clones disappeared on passage in culture. This antigen was not expressed at all by the avirulent iir vitro-derived line prepared with cells from the same calf Both antigens were expressed by lines infected with other stocks of T. annulata, including two lines known to induce lethal disease. The different profiles of expression of the two novel antigens, recognized by MAbs EU1 and EU106, by the line undergoing attenuation suggest (1) that the two antigens interact differently with the bovine immune system; and (2) that there are two, very different, potential roles for these antibodies in the development of vaccines against T. annulata infections.
Till D D, Linz B, Seago J E, Elgar S J, Marujo P E, Elias M D, Arraiano C M, McClellan J A, McCarthy J E G, Newbury S F (1998)

Identification and developmental expression of a 5 '-3 ' exoribonuclease from Drosophila melanogaster

Mechanisms of Development 79 (1-2), 51-55


In multicellular organisms, very little is known about the role of mRNA stability in development, and few proteins involved in degradation pathways have been characterized. We have identified the Drosophila homologue of XRN1, which is the major cytoplasmic 5'-3' exoribonuclease in Saccharomyces cerevisiae. The protein sequence of this homologue (pacman) has 59% identity to S. cerevisiae XRN1 and 67% identity to the mouse homologue (mXRN1p) in certain regions. Sequencing of this cDNA revealed that it includes a trinucleotide repeat (CAG)(9) which encodes polyglutamine. By directly measuring pacman exoribonuclease activity in yeast, we demonstrate that pacman can complement the yeast XRN1 mutation. Northern blots show a single transcript of approximately 5.2 kb which is abundant only in 0-8-h embryos and in adult males and females. In situ hybridization analysis revealed that the pcm transcripts are maternally derived, and are expressed at high levels in nurse cells. During early embryonic syncytial nuclear divisions, pcm transcripts are homogenously distributed. pcm mRNA is expressed abundantly and ubiquitously throughout the embryo during gastrulation, with high levels in the germ band and head structures. After germ band retraction, pcm transcripts are present at much lower levels: in agreement with the Northern results. Our experiments provide the first example of an exoribonuclease which is differentially expressed throughout development.
Anderson G, Anderson K L, Tchilian E Z, Owen J J T, Jenkinson E J (1997)

Fibroblast dependency during early thymocyte development maps to the CD25(+) CD44(+) stage and involves interactions with fibroblast matrix molecules

European Journal of Immunology 27 (5), 1200-1206


We have investigated the role of specific components of the thymic stroma during development of CD4(-)8(-) T cell precursors by separating and reaggregating precursor subsets with individual or combinations of stromal cells. We show that while the development of CD25(+) 44(+) precursors is dependent upon a combination of major histocompatibility complex (MHC) class II+ thymic epithelial cells and fibroblasts, their direct descendants, CD25(+) 44(-) precursors, develop to the CD4(+) 8(+) stage in the presence of MHC class II+ thymic epithelial cells alone. Thus, CD25(+) 44(+) precursors are the last developmental stage to be dependent upon fibroblast support. In addition, while metabolically inactive, 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (ECDI)-treated fibroblasts retain the ability to promote T cell development, prior treatment with hyaluronidase abrogates this effect, suggesting that fibroblast-associated extracellular matrix components are the key elements involved. In support of this, we show that fibroblasts are located in cortical regions of the thymus where T cell precursors are known to reside, and that these fibroblasts are associated with an extensive extracellular matrix not found on thymic epithelial cells. Finally, antibodies to alpha 4 integrin and CD44 interfere with the efficiency with which CD4(+) 8(+) cells are generated from CD25(+) 44(+) precursors in reaggregate cultures and also reduce the binding of the latter to 3T3 fibroblasts, suggesting these molecules play a role in bringing T cell precursors into contact with fibroblast-associated extracellular matrix.
Cabrita G, Iqbal M, Reddy H, Kemp G (1997)

Activation of the adenovirus protease requires sequence elements from both ends of the activating peptide

Journal of Biological Chemistry 272 (9), 5635-5639


The adenovirus protease requires activation by an 11-residue peptide, GVQSLKRRRCF, to achieve maximum proteolytic activity. Derived from the C terminus of the viral protein pVI, the activating peptide (pVI-CT) forms a disulfide bond with cysteine 104 of the protease and causes a conformational change that accompanies the development of proteolytic activity, Results presented here show that the interaction of pVI-CT with the protease is dependent not only on the cysteine 10 but also on glycine 1 and valine 2, Removal of these residues, acetylation of the N-terminal glycine, or mutation of the valine to alanine or threonine significantly reduces or abolishes activation, Peptides lacking Gly-l and Val-2 still form a disulfide bond with the protease but do not cause a conformational change in the protease also they are not effective inhibitors of activation as the interaction is readily reversed by full-length pVI-CT. These results suggest that pVI-CT causes activation by binding to two distinct regions of the protease and in doing so stabilizes the catalytic site, The reversible nature of the activation, suggested by the results presented here, may well reflect an in vivo regulatory mechanism.
Tchilian E Z, Owen J J T, Jenkinson E J (1997)

Anti-alpha 4 integrin antibody induces apoptosis in murine thymocytes and staphylococcal enterotoxin B-activated lymph node T cells

Immunology 92 (3), 321-327


We have shown that an antibody (9C10) to the alpha 4 integrin induces apoptosis in murine immature CD4(+) CD8(+) thymocytes and in activated (but not resting) mature lymph node T cells. In both cases, apoptosis is blocked by the highly selective protein kinase C (PKC) inhibitor Ro31.8425, suggesting that 9C10 induces signalling through the alpha 4 integrin resulting in PKC activation leading to apoptosis. Overall, our results indicate the potential role of the alpha 4 integrin-mediated interactions in apoptosis induction during T-cell development and following mature T-cell activation.
Jones S J, Iqbal M, Grierson A W, Kemp G (1996)

Activation of the protease from human adenovirus type 2 is accompanied by a conformational change that is dependent on cysteine-104

Journal of General Virology 77 (8), 1821-1824


Adenovirus codes for a protease the activity of which can be regulated in vitro by an 11 residue peptide (GVQSLKRRRCF) derived from another viral protein, pVI. Three cysteine residues, one in the activating peptide and two in the protease (C104 and C122), play a central role in both activation and catalysis. Expression of protease mutants in insect cells has shown that C104 is not essential for proteolytic activity. GVQSLKRRRCF also caused a concentration-dependent increase in tryptophan fluorescence of protease expressed in Escherichia coli that paralleled the increase in proteolytic activity, indicating that activation was accompanied by a conformational change. Tryptophan fluorescence of C104S was not increased by the addition of GVQSLKRRRCF, nor was the fluorescence of wildtype protease increased by the addition of the peptide analogues where cysteine is replaced by aspartic acid or serine, suggesting that C104 is involved in activation and C122 in catalysis.
Knight P, Musoke A J, Gachanja J N, Nene V, Katzer F, Boulter N, Hall R, Brown C G D, Williamson S, Kirvar E, Bell-Sakyi L, Hussain K (1996)

Conservation of neutralizing determinants between the sporozoite surface antigens of Theileria annulata and Theileria parva

Experimental Parasitology 82 (3), 229-241


The sporozoite surface antigens SPAG-1 of Theileria annulata and p67 of Theileria parva are postulated to contain determinants necessary for host cell invasion and/or recognition and are both being considered as candidates for inclusion in subunit vaccines. Preliminary data suggest that these are related molecules. In this paper we describe the investigation of the relationship between these sporozoite antigens further by analysis of the immunological cross-reactivity using Mabs and sera raised against each antigen. The cross-reactions were examined by carrying out Western blots, IFA tests, and in vitro sporozoite neutralization assays. In addition, sequence comparison data which clearly establish that these surface antigens are encoded by related genes are presented. The regions of SPAG-1 identified as containing cross-reactive epitopes recognized by p67 antiserum correlated to regions of high predicted homology between p67 and SPAG-1, which are located at their respective N- and C-termini. Furthermore, p67 and SPAG-1 were found to contain cross-reactive determinants responsible for neutralization of sporozoite infectivity in vitro, and at least some of these were located in the C-termini of both molecules. The relevance of these findings to the-possible roles for these molecules in host cell invasion is discussed. (C) 1996 Academic Press, Inc.
Mercer D K, Iqbal M, Miller P G G, McCarthy A J (1996)

Screening actinomycetes for extracellular peroxidase activity

Applied and Environmental Microbiology 62 (6), 2186-2190


A diverse collection of actinomycete strains were screened for production of extracellular peroxidase activity by adapting a chemiluminescence analysis system developed for horseradish peroxidase-based enzyme-linked immunosorbent assay. Extracellular peroxidase activity was found to be common but quantitatively variable, and this rapid and sensitive screening system permitted identification of a small group of high-producing strains, A range of spectrophotometric assays were compared for the measurement of peroxidase activity in concentrated culture supernatants of two selected thermophilic streptomycetes, Of these, the peroxide-dependent oxidation of 2,4-dichlorophenol was identified as the most robust and reproducible assay for quantitative studies.
Tchilian E Z, Anderson G, Moore N C, Owen J J T, Jenkinson E J (1996)

Involvement of LFA-1/ICAM-2 adhesive interactions and PKC in activation-induced cell death following SEB rechallenge

Immunology 87 (4), 566-572


Ligation of T-cell receptor (TCR) causes mature T cells to proliferate or, on re-exposure to antigen, can cause them to die by activation-induced cell death (AICD). In proliferative responses, costimulatory and adhesive interactions are required and activation of protein kinase C (PKC) has been shown to be essential. Whether or not interactions involving costimulatory signals and PKC have a role in facilitating AICD remains unclear. Here we have examined the role of CD28/B7 and leucocyte function associated antigen-1 (LFA-1)/intracellular adhesion molecule (ICAM) mediated interactions in AICD triggered by staphylococcal enterotoxin B (SEE) in murine lymph node T cells. We show that, after a primary proliferative response to SEE, LFA-1/ICAM-2 adhesive interactions can play a part in AICD following SEE rechallenge, while B7 and ICAM-1 mediated interactions are not essential for this process. In addition, using a highly selective PKC inhibitor, Ro31.8425, we show that PKC activation is essential for the regulation of AICD by SEE rechallenge.


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