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By immunizing prion knockout mice (Prnp-/-) with recombinant murine prion protein (PrP(c)), we obtained a panel of mAbs specific for murine PrP(c). These mAbs can be applied to immunoblotting, cell surface immunofluorescent staining, and immunohistochemistry at light and electron microscopy. These mAbs recognize both the normal (PrP(c)) and protease-resistant (PrP(res)) isoforms of PrP. Some mAbs are species restricted, while others react with PrP from a broad range of mammals including mice, humans, monkeys, cows, sheep, squirrels, and hamsters. Moreover, some of the mAbs selectively recognize different PrP glycoforms as well as the metabolic fragments of PrP(C). These newly generated PrP(C) antibodies will help to explore the biology of PrP(c) and to establish the diagnosis of prion diseases in both humans and animals

We previously demonstrated that recombinant plant virus particles containing a chimeric peptide representing two rabies virus epitopes stimulate virus neutralizing antibody synthesis in immunized mice. We show here that mice immunized intraperitoneally or orally (by gastric intubation or by feeding on virus-infected spinach leaves) with engineered plant virus particles containing rabies antigen mount a local and systemic immune response. After the third dose of antigen, given intraperitoneally, 40% of the mice were protected against challenge infection with a lethal dose of rabies virus. Oral administration of the antigen stimulated serum IgG and IgA synthesis and ameliorated the clinical signs caused by intranasal infection with an attenuated rabies virus strain.

Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet--which is also a widespread motif involved in cell-to-cell adhesion--can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs, can contribute to phenotypic flexibility of RNA viruses in nature

The immunoreactivity and allergenicity of proteins present in processed food (UHT milk, yoghurt, hard cheese, cottage cheese, biscuit, and sausage intended for children) were determined in this study. Proteins were characterised by SDS-PAGE electrophoresis. The changes in immunoreactivity were compared by enzyme-linked immunosorbent assay (ELISA) using polyclonal rabbit antibodies specific to alpha-lactalbumin, beta-lactoglobulin, alpha-, beta-, and kappa-casein. The allergenicity was determined with human pooled sera from CMA allergic patients by ELISA and immunoblot. The results have shown that the allergenicity of the food products is mainly correlated with bovine serum albumin (BSA), lactofferin (LF), and alpha-casein or the products of non-specific reactions between carbohydrate and proteins (e.g. lactosylation).

A cDNA representing the plastic-encoded homolog of the prokaryotic ATP-dependent protease ClpP was amplified by reverse transcription-polymerase chain reaction, cloned, and sequenced. ClpP and a previously isolated cDNA designated ClpC, encoding an ATPase related to proteins encoded by the ClpA/B gene family, were expressed in Escherichia coli. Antibodies directed against these recombinant proteins recognized proteins in a wide variety of organisms. N-terminal analysis of the Clp protein isolated from crude leaf extracts showed that the N-terminal methionine is absent from ClpP and that the transit peptide is cleaved from ClpC. A combination of chloroplast subfractionation and immunolocalization showed that in Arabidopsis, ClpP and ClpC localize to the stroma of the plastid. Immunoblot analyses indicated that ClpP and ClpC are constitutively expressed in all tissues of Arabidopsis at levels equivalent to those of E. coli ClpP and ClpA, ClpP, immunopurified from tobacco extracts, hydrolyzed N-succinyl-Leu-Tyr-amidomethylcoumarin, a substrate of E. coli ClpP. Purified recombinant ClpC facilitated the degradation of 3H-methylcasein by E. coli ClpP in an ATP-dependent fashion. This demonstrates that ClpC is a functional homolog of E. coli ClpA and not of ClpB or ClpX. These data represent the only in vitro demonstration of the activity of a specific ATP-dependent chloroplast protease reported to date

We have characterized the electrophoresis profile of bovine serum conglutinin and used Western blotting to compare profiles of this lectin derived from the sera of different breeds of cattle. The profile of non-reduced conglutinin is characterised by many bands with molecular masses ranging from 34 to 630 kDa. Reduced lectin takes the form of three main bands with molecular masses of 41, 47 and 96 kDa. We show that conglutinin is present not only in adult bovine serum, but also in foetal bovine serum, colostrum and milk. The sera of sheep, goats, gnu antelopes and deer, as well as some non-ruminant species such as llamas, horses, boars, pigs and humans, contain proteins which have similar antigenicity to that of bovine conglutinin. These reacted with monoclonal and polyclonal antibodies specific for bovine conglutinin under reducing and non-reducing conditions in Western blotting. The protein profiles of bison and swine lectin were observed to be particularly similar to bovine conglutinin.

Cutaneous epitheliotropic T-cell lymphoma with progression to superficial and internal lymph nodes and the spleen was diagnosed in a two-year-old Holstein-Friesian cow. The skin lesions included multiple hypotrichous to alopecic nodules, often with ulceration, which first appeared three months after calving and progressed quickly to cover the entire body. The cow was euthanized one month later. The ELISA test excluded bovine leukaemia virus infection. Histologically, the formation of Pautrier's microabscesses was observed in the epidermis and dense neoplastic infiltration with mild folliculotropism was observed in the dermis. Similar neoplastic cells were present in the histological sections of lymph nodes and spleen. Immunohistochemical analysis was performed using CD3, CD79alphacy, HLA-DR, WC1-N3 and Ki67 antibodies. Immunophenotyping results (CD79alphacy-, CD3+, WC1-N3-) confirmed the alphabeta T-cell origin of neoplastic cells. The mean Ki67 index among neoplastic cells was 15.3%. On the basis of the immunohistochemical and histopathological results, the first case in Poland of cutaneous epitheliotropic T-cell lymphoma in a cow was confirmed. Additionally, MHC class II expression on approximately 10.4% of lymphoma cells was associated with a poor clinical prognosis. However, the up-regulation of MHC class II expression on accompanying cells suggested tumour immune surveillance, an antigen-specific immune response or immunosuppression.

Peptides corresponding to the immunodominant loop located at residues 135-158 on capsid protein VP1 of foot-and-mouth disease virus (FMDV) generally elicit high levels of anti-peptide and virus-neutralizing antibodies. In some instances, however, the level of neutralizing antibodies is low or even negligible, even though the level of anti-peptide antibodies is high. We have shown previously that the antigenic activity of peptide 141-159 of VP1 of a variant of serotype A can be mimicked by a retro-inverso (all-D retro or retroenantio) peptide analogue. This retro-inverso analogue induced greater and longer-lasting antibody titers than did the corresponding L-peptide. We now show that a single inoculation of the retro-inverso analogue elicits high levels of neutralizing antibodies that persist longer than those induced against the corresponding L-peptide and confer substantial protection in guinea pigs challenged with the cognate virus. In view of the high stability to proteases of retro-inverso peptide analogues and their enhanced immunogenicity, these results have practical relevance in designing potential peptide vaccines

A method for purification of carp serum immunoglobulin (IgM), intended for the production of monoclonal antibodies, is described in the present study. Hybridomas that produce antibodies against IgM heavy chain were selected by ELISA and Western blotting. Ascitic fluids were prepared and tested by the above mentioned methods, followed by their typing. Monoclonal antibody with the highest titre of antibodies against carp immunoglobulin was selected for conjugation with horseradish peroxidase. Specificity of conjugated monoclonal antibody was tested in a panel of various fish species sera. Cross-reactivity was not detected in 12 fish species, including Oncorhynchus mykiss. Besides Cyprinus carpio, positive results were also found in Carassius auratus, Aristichthys nobilis and Silurus glanis. The sensitivity in common carp was approximately 10 ng/mL.

The N-demethylation of the pyridazinone pro-herbicide metflurazon into norflurazon implies a toxification in photosynthetic organisms. This is confirmed by quantitative structure activity relationships determined for two unicellular green algae, Chlorella sorokiniana and Chlorella fusca; however, the latter is 25 to 80 times more sensitive to metflurazon. This sensitivity is linked to differences in the N-demethylase activity of both algae, as determined by an optimized in vivo biotransformation assay. Apparent Km values of the metflurazon-N-demethylase indicate a 10-fold higher affinity for this xenobiotic substrate for Chlorella fusca. Furthermore, algal metflurazon-N-demethylation is characterized by distinct variations in activity, depending on the stage of cell development within the cell cycle. Several well-established inhibitors of cytochrome P450-mediated reactions, including piperonylbutoxide, 1-aminobenzotriazole, 1-phenoxy-3-(1H-1,2,4-triol-1yl)4-hydroxy-5,5-di methylhexane, and tetcyclacis, as well as cinnamic acid, a potential endogenous substrate, inhibited the N-demethylation of metflurazon. The results suggest that the N-demethylation of metflurazon by both algae is mediated by a cytochrome P450 monooxygenase. The determination of antigenic cross-reactivity of algal proteins with heterologous polyclonal antibodies originally raised against plant P450s, anti-cinnamic acid 4-hydroxylase (CYP73A1), anti-ethoxycoumarin-O-dealkylase, anti-tulip allene oxidase (CYP74), and an avocado P450 (CYP71A1) or those of bacterial origin, CYP105A1 and CYP105B1, suggests the presence of distinct P450 isoforms in both algae