Evidence for phospholipase B activity in Fusobacterium necrophorum cultures and its association with hemolysin/leucocidin activities.

Phospholipase B (PLB) activity was present in Fusobacterium necrophorum cultures and it correlated closely with virulence and co-purified with the hemolysin/leucocidin activities. All three activities were associated with a large molecule or molecular complex (6 x 10(2)-2 x 10(3) kDa) exhibiting varying degrees of aggregation. These were present mainly in the culture medium and to a lesser extent in cell sonicates. The PLB and toxin activities were sensitive to heat, dissociating agents, proteolytic enzymes, prolonged purification regimes, freeze-drying and repeated freeze-thawing. The toxin(s) were stable over a broad range of pH, did not require divalent ions or reducing agents and could be kept for several months as an ammonium sulfate precipitate at 4 degrees C, or stored as a concentrated liquid in the presence of proteolytic inhibitors at - 20 degrees C.

Soluble Mycobacterium bovis protein antigens: studies on their purification and immunological evaluation.

The eradication of bovine tuberculosis is an ultimate aim of the beef industry and the development of accurate diagnostic tests will expedite eradication. Characterization of Mycobacterium bovis antigens, and a detailed understanding of their immune reactivity will aid in the development of more specific and sensitive diagnostic tests. With this aim, studies were conducted which have resulted in the purification and immunological characterization of the major soluble M. bovis antigens. The purified antigens were found to contain cross-reactive epitopes and immunological responses to these proteins varied among individual animals. Thus if more specific diagnostic tests are to be formulated, they will have to be at the epitope level, using only species-specific epitopes and not whole proteins. Due to the genetic diversity of the response of infected cattle to individual epitopes, a large cocktail of such epitopes will be necessary for the development of a sensitive test.

Serological reactivity to Mycobacterium bovis protein antigens in cattle.

The serological response to 12 purified Mycobacterium bovis antigens were examined in an ELISA assay. These antigens included the majority of M. bovis protein antigens described to date and in most cases they were very similar to the M. tuberculosis antigens of the same molecular mass. The purified antigens were tested against sera from M. bovis infected cattle, M. bovis culture-negative cattle from infected herds and animals infected with related microorganisms, mainly other mycobacterial species. All the antigens gave strong reactions with at least some sera from the M. bovis infected group and showed cross-reactivity with some of the sera from the other two groups. The antigen with the highest specificity reacted strongly with only 60% of the M. bovis infected sera. Antigens that reacted with most or all of the M. bovis infected sera also gave the highest cross-reactivity with sera from the other two groups. These results indicate that a serological test based on any one or a combination of these antigens, without removal of the cross-reacting epitopes, would be unsatisfactory.

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis.

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigen in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K. A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.

A field evaluation of serological and cellular diagnostic tests for bovine tuberculosis.

This paper describes the field evaluation of a serological test and a new in vitro assay for cell-mediated reactivity for the diagnosis of bovine tuberculosis. The use of a Mycobacterium bovis-specific antigen (MPB-70) in an ELISA to test the serological response to tuberculosis infection resulted in a specificity of 96.4% and a sensitivity of 18.1%. The most favourable results were obtained with the interferon gamma (IFN-gamma) assay which had a sensitivity of 81.8% and a specificity of 99.1%. Respective figures for the single intradermal tuberculin test were 68.1% and 96.7%. The use of MPB-70 as the antigen in the IFN-gamma assay reduced the sensitivity of this assay, without producing any useful increase in specificity. The IFN-gamma assay was also demonstrated to be a practical diagnostic test for use with large groups of cattle.

Heat stress induces epithelial plasticity and cell migration independent of heat shock factor 1.

Current cancer therapies including cytotoxic chemotherapy, radiation and hyperthermic therapy induce acute proteotoxic stress in tumour cells. A major challenge to cancer therapeutic efficacy is the recurrence of therapy-resistant tumours and how to overcome their emergence. The current study examines the concept that tumour cell exposure to acute proteotoxic stress results in the acquisition of a more advanced and aggressive cancer cell phenotype. Specifically, we determined whether heat stress resulted in an epithelial-to-mesenchymal transition (EMT) and/or the enhancement of cell migration, components of an advanced and therapeutically resistant cancer phenotype. We identified that heat stress enhanced cell migration in both the lung A549, and breast MDA-MB-468 human adenocarcinoma cell lines, with A549 cells also undergoing a partial EMT. Moreover, in an in vivo model of thermally ablated liver metastases of the mouse colorectal MoCR cell line, immunohistological analysis of classical EMT markers demonstrated a shift to a more mesenchymal phenotype in the surviving tumour fraction, further demonstrating that thermal stress can induce epithelial plasticity. To identify a mechanism by which thermal stress modulates epithelial plasticity, we examined whether the major transcriptional regulator of the heat shock response, heat shock factor 1 (HSF1), was a required component. Knockdown of HSF1 in the A549 model did not prevent the associated morphological changes or enhanced migratory profile of heat stressed cells. Therefore, this study provides evidence that heat stress significantly impacts upon cancer cell epithelial plasticity and the migratory phenotype independent of HSF1. These findings further our understanding of novel biological downstream effects of heat stress and their potential independence from the classical heat shock pathway.

Purification of 3-phosphoglycerate kinase from diverse sources by affinity elution chromatography.

1. Affinity elution chromatography was used to purify phosphoglycerate kinase from a variety of sources. The choice of buffer pH for the chromatography was made according to the relative electrophoretic mobility of the enzyme from the species concerned. 2. Outlines of the methods used to isolate the enzyme from over 20 sources are presented. The enzyme was purified from the muscle tissue of a variety of mammals, fish and birds, from liver of several animals, from yeast, Escherichia coli, and plant leaves. The more acidic varieties of the enzymes were purified by conventional gradient elution from ion-exchangers as affinity elution procedures were not applicable. 3. The structural and kinetic parameters investigated show that phosphoglycerate kinase is evolutionarily a highly conservative enzyme; there were few differences in properties regardless of source or function (glycolytic, gluconeogenic or photosynthetic). 4. A detailed comparison of the enzyme preparations purified from bovine muscle and bovine liver failed to detect any significant differences between them; the evidence indicates that they are genetically identical.

Cellular and humoral immune responses of cattle to purified Mycobacterium bovis antigens.

Cellular responses to several purified antigens of Mycobacterium bovis were examined in experimentally infected cattle over a period of 36 months, using in vitro cellular proliferation and interferon-gamma assays. These antigens (12, 19, 22a, b, 24, 25, 30, 32, 39, 65 and 70 kDa) included the majority of M. bovis protein antigens described to date and are highly homologous to those purified from M. tuberculosis. Cellular responses were examined at 3-month time intervals during the 36-month course of infection. All purified antigens induced cellular immune responses in the infected animals. The onset and magnitude of response to individual antigens varied among the animals. At any specific time during the period of infection one or more antigens appeared to be immunodominant but the immunodominance profile changed as the infection progressed. Humoral immune responses were low or absent in the first half of the infection period, but increased substantially for some of the antigens during the second half. Variation was observed among the different animals as to which antigens they recognized.

Purification and characterization of major antigens from a Mycobacterium bovis culture filtrate.

Ten major antigens from Mycobacterium bovis culture filtrate of 39, 32, 30, 25, 24, 22 (a and b forms), 19, 15, and 12 kDa have been purified and characterized by classical physicochemical methods. With monoclonal antibodies and/or N-terminal amino acid sequencing data, it was found that the antigens of 32, 30, 24, 22 (a), 19, and 12 kDa are related to M. bovis or M. tuberculosis antigens P32, MPB59, MPB64, MPB70, 19 kDa, and 12 kDa, respectively. The 39-, 25-, 22 (b)-, and 19-kDa antigens showed concanavalin A-binding properties and were positive in a glycan detection test, suggesting that they are glycoproteins. The 25- and 22 (b)-kDa proteins were found to be glycosylated forms of MPB70.

Molecular species of epidermal growth factor carrying immunosuppressive activity.

The suppression of antibody formation to sheep red cells in mice by partially purified fractions of mouse submaxillary gland was shown to be caused by epidermal growth factor (EGF). Purification of EGF by the method of Savage and Cohen resolved three components referred to as EGF a, EGF b, and EGF c. All three induced premature eye opening in neonatal mice, but only EGF a (identified as EGF 1-53) had full immunosuppressive activity. EGF c was shown by micropeptide mapping of chymotryptic and thermolytic digests and amino-terminal analysis to differ from EGF a only by the presence of beta-aspartyl instead of an asparaginyl residue. EGF b differed from EGF a in that it lacked the N-terminal asparagine. EGF shortened enzymatically at its carboxy terminal by two or five amino acids did not have any immunosuppressive activity. These findings suggest that, in contrast to some other biological effects of EGF, intact amino and carboxy terminals are required for the expression of immunosuppressive activity.

Purification of a major Mycobacterium bovis antigen for the diagnosis of bovine tuberculosis.

A Mycobacterium bovis antigen has been purified from culture filtrate by chromatofocusing. This antigen is a major component of culture filtrate and cell extracts and shows a considerable degree of micro-heterogeneity in electric charge and molecular weight. Studies with monoclonal and polyclonal antibodies raised against the purified antigen show that some of its antigenic determinants also occur in higher molecular weight species in culture filtrate and particularly in whole cell preparations. Immunoblotting and ELISA studies, using sera from M.bovis-infected animals, showed that this antigen is one of the most immunoreactive components of M. bovis, recognized by the majority of animals with detectable antibody response to M. bovis. The specificity of the purified antigen is far superior to that of the crude culture filtrate, with very few false positive results. The purified antigen also elicits strong in vivo and in vitro cell-mediated responses. The amino acid compositions of two variants of this antigen have been determined and found to be similar to that of MPB-70.

Differential T cell responses to mycobacteria-secreted proteins distinguish vaccination with bacille Calmette-Guérin from infection with Mycobacterium tuberculosis.

The immune responses of healthy recipients of Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine, tuberculosis (TB) patients, and contacts of TB patients were examined to three major secretory proteins of Mycobacterium tuberculosis, MPB59, MPB64, and MPB70. MPB59 evoked a T cell response in 78% of BCG vaccines, 62% of TB patients, and 60% of contacts. MPB64 and MPB70 were recognized by < 15% of BCG vaccinees, half of TB patients, and three-quarters of contacts. TB and leprosy patients had antibody responses to MPB59, but few had antibodies to MPB64 or MPB70. Hybridization of mycobacterial DNA with specific gene probes demonstrated the absence of a gene for MBP64 in the vaccine strain of BCG, but the MPB70 gene was found in all virulent and vaccine BCG strains tested. Since MPB64 and MPB70 can induce delayed-type hypersensitivity reactions in infected animals, either of these proteins may have potential as skin test reagents for detecting infection with M. tuberculosis.

Bands, interbands and puffs in native Drosophila polytene chromosomes are recognized by a monoclonal antibody to an epitope in the carboxy-terminal tail of histone H1.

A monoclonal antibody was raised against Drosophila melanogaster histone H1. Immunoscreening of proteolytic cleavage fragments of H1 and of a set of all possible overlapping synthetic octapeptides corresponding to the amino acid sequence of H1, revealed that the antibody recognizes an epitope within the sequence 207VTAAKPKA214 near the centre of the carboxy-terminal tail. This antibody gives positive immunofluorescence over the entire length of native D. melanogaster polytene chromosomes isolated from salivary glands by microdissection at physiological pH and ionic strength. Bands, interbands and puffs are all seen to contain H1. The immunofluorescence over puffs, albeit lower than that over bands and interbands, indicates that chromatin decondensation can occur without complete loss of H1 in these structures. The reaction of the antibody with bands suggests that the segment of the C-terminal tail containing the epitope may be exposed in the condensed 30 nm chromatin filament.