Publications

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

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Abstract

PCR analysis of the genomes of 18 different African swine fever virus (ASFV) isolates showed that the I14L open reading frame (ORF) was present as either a long form or short form in all of the isolates. Sequencing of the ORF from eight isolates confirmed that both forms of the ORF were well conserved. Antisera raised against the I14L protein identified the long form of the protein as a 21 kDa protein expressed late during ASFV infection. Immunofluorescent analysis of transiently expressed haemagglutinin-tagged forms of the I14L protein showed that the long form of the protein localized predominantly to the nucleus and within the nucleoli. In contrast, although the short form of the protein was also present predominantly in the nucleus, it did not localize to the nucleoli. Deletion of the N-terminal 14 amino acids from the long form of the I14L protein, which includes a high proportion of basic Arg/Lys residues, abolished the specific nucleolar localization of the protein, although the protein was still present in the nucleus. Addition of this 14 amino acid sequence to beta-galactosidase or replacement of the N-terminal 14 amino acids of the I14L short form with those from the long form directed both of these modified proteins to the nucleolus. This indicates that this 14 amino acid sequence contains all the signals required for nucleolar localization.
Montoya M, Del Val M (1999)

Intracellular rate-limiting steps in MHC class I antigen processing

Journal of Immunology 163 (4), 1914-1922

Abstract

Quantitative aspects of the endogenous pathway of Ag processing and presentation by MHC class I molecules to CB8(+) CTL were analyzed over a wide range of Ag expression in recombinant vaccinia virus-infected cells expressing beta-galactosidase as model Ag, Only the amount of starting Ag was varied, leaving other factors unaltered, Below a certain level of Ag synthesis, increasing protein amounts led to a sharp rise in recognition by CTL, Higher levels of Ag expression led to a saturation point, which intracellularly limited the number of naturally processed peptides bound to MHC and thereby also CTL recognition, The rate-limiting step was located at the binding of the antigenic peptide to MHC inside the vaccinia virus-infected cell or before this event.

Abstract

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).

Abstract

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

Abstract

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.

Abstract

A clinical trial testing the prophylactic effect of a 5 mg kg(-1) dose of buparvaquone on either Theileria annulata or Theileria parva experimental infections of calves demonstrated its efficacy for periods of at least seven days. The drug given 1 h or seven days before 50% lethal T. annulata sporozoite infection protected all eight calves, but prophylaxis was insufficient after 14 days to protect two out of four calves from severe reaction. When immunity was challenged by a lethal second parasite dose a month after the first, all these calves were immune. In the ir: parva trial, calves given drug 1 h or seven days before a 25% lethal infection underwent minimal reactions, but some were over-protected and were susceptible to a similar challenge sporozoite dose. Although drug levels remaining 14 days after prophylaxis protected these calves from the mild challenge, some parameters measured were within the range of the 'no drug' control group. These results indicate the effectiveness of a single 5 mg kg(-1) dose of buparvaquone for more than seven days but also the potential risk of its use in the infection and treatment method of immunisation. It is suggested that there may be circumstances where simple field prophylactic treatment with buparvaquone may be beneficial.

Abstract

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

Abstract

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.

Abstract

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.

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