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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.


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.


We have developed an in vitro system in which Staphylococcal enterotoxin B (SEB) activated murine lymph node T cells undergo apoptosis following a rechallenge with SEB. Approximately 50% of the cell are induced to die following SEB rechallenge and this apoptosis is sensitive to the PKC inhibitor Ro3 1.8425 and cyclosporin A. We have also found that cells, previously activated with SEB, can be induced to undergo apoptosis with an anti-a4 mAb (9C10) in the absence of any hrther exposure to antigen. Apoptosis induced in these cells by anti-a4 mAb is inhibited by cyclosporin A and Ro3 1.8425, in a comparable way to restimulation with SEB. We suggest that VLA-4 acitvation may be an important costimulatory factor in the induction of apoptosis in activated mature T cells. This pathway may represent one of the mechanisms whereby certain integrin-cell/ECM interactions can regulate apoptosis in activated mature T cells.


A C terminal fragment (SR1) of SPAG-1, a sporozoite surface antigen of Theileria annulata, has been expressed as a fusion protein in the el loop of hepatitis B cope antigen (HBcAg), This recombinant antigen (HBcAg-SR1) is produced in the form of self-assembling polyhedral particles which have been visualised under the electron microscope, Cattle immunised with HBcAg-SR1 produced high titres of neutralising antibodies, A significant T cell response to both the HBcAg and SR1 determinants was observed but evidence of a T suppressor determinant in SR1 was also revealed Immunised cattle showed some evidence of protection to sporozoite challenge as assessed by severity of the disease. The significance of these findings for the development of a sub-unit vaccine against T, annulata is discussed.


Embryonated eggs were coinfected with two strains of the coronavirus avian infectious bronchitis virus (IBV), IBV-Beaudette and IBV-M41, to investigate whether recombination between the two strains would occur. Virions were isolated from the allantoic fluid of the coinfected eggs and putative hybrid RNAs were detected by polymerase chain reaction (PCR), using strain-specific oligonucleotides. PCR products, of the expected sizes, were obtained as predicted from potential recombination events between the nucleoprotein (N) gene and the 3'-untranslated region of the two IBV genomes. Sequencing confirmed that they corresponded to hybrid RNAs. Virus produced as a result of the mixed infection was treated with an M41-specific neutralizing monoclonal antibody and passaged in Vero cells, in which IBV-Beaudette, but not IBV-M41, replicated. Hybrid RNA was still detectable after three serial passages. Since no IBV-M41 was detectable this confirmed that infectious recombinant genomes had been produced in the embryonated eggs. These findings not only support the circumstantial evidence, from sequencing studies of IBV field strains, that recombination occurs during replication of IBV and contributes to the diversity of IBV, but also show that coronavirus RNA recombination is not limited to mouse hepatitis virus.
Tchilian E Z, Anderson G, Moore N C, Owen J J, Jenkinson E J (1995)

Effects of activation on the regulation of apoptosis in lymph node T cells

Journal of Cellular Biochemistry Supplement (19B), 325
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Nitric oxide (NO) was produced when bovine peripheral blood mononuclear cells (PBMC) or purified, adherent PBMC (macrophages) were incubated in vitro with bovine recombinant interferon gamma (Bo rIFN-?). NO was produced by cells from naive, uninfected calves as well as by cells from cattle either infected with or recovered from infection with Theileria annulata or Theileria parva. PBMC of cattle undergoing tropical theileriosis (T. annulata infection) or East Coast fever (T. parva infection) synthesized NO spontaneously in vitro. NO was also induced when PBMC of immune, but not of naive, cattle were cultured with T. annulata macroschizont-infected cell lines. Macrophages alone were not stimulated to produce NO by such infected cells. In vitro establishment of macroschizont-infected cell lines was suppressed either by incubating sporozoites with S-nitroso-N-acetyl-DL-penicillamine (SNAP), a NO releasing molecule, prior to invasion of PBMC or by pulsing developing cultures of trophozoite-infected cells with SNAP. Proliferation of established macroschizont-infected cell lines was not affected by SNAP. Taken together with the well documented roles of NO in neurotransmission, vasodilatation, cell and tissue damage and immunosuppression, the results presented here indicate that NO may not only protect cattle against T. annulata and T. parva but, if produced in excess, play a prominent role in the pathogenesis of tropical theileriosis and East Coast fever.


Streptomyces thermoviolaceus is a thermophilic actinomycete that was found to produce relatively large amounts of extracellular peroxidase activity when grown on xylan as primary carbon source. The activity was due to multiple isoforms of peroxidase, of which two, designated P-3 and P-5, were predominant. The two proteins were purified to homogeneity by a combination of ultrafiltration, ammonium sulphate precipitation, anion-exchange chromatography, gel filtration and preparative gel electrophoresis. The peroxidases were found to be haemoproteins that catalysed the oxidation of a range of substrates in the presence of hydrogen peroxide. Both are monomeric acidic proteins (P-3: 82 kDa, pl 5.0; P-5: 60 kDa, pl 4.75) but with some differences in substrate specificity, P-3 exhibiting the broader substrate range. Peroxidase activity was optimal at ph values close to neutrality, and both enzymes were robust, exhibiting activity at elevated temperatures in the presence of denaturing agents such as SDS or 8 M urea. Peroxidase P-3 was stable at 50 degrees C for more than 24 h and had a half-life of 70 min at 70 degrees C. Polyclonal antibodies prepared against each isoform cross-reacted, indicating that the proteins were antigenically related. No cross-reactions were detected against horseradish peroxidase or crude peroxidase preparations from two other thermophilic streptomycetes.


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