Association of tumor-associated antigen on the proliferation of bovine leukemia virus-infected lymphoblastoid B-cell lines.

The association of tumor-associated antigen (TAA) on the proliferation of BLV-infected lymphoblastoid B-cell lines (BL2M3 and BL312) was investigated. Flow cytometric analysis of the expression of TAA with monoclonal antibody (mAb) c143 showed high expression of TAA on the surfaces of BL2M3 and BL312 cells. A large amount of TAA was found in the culture supernatant of BL2M3 and BL312 cells as well as in the lysates of BL2M3 and BL312 cells. Culture supernatant but not lysates of BL2M3 and BL312 cells promoted the growth of either BL2M3 cells or BL312 cells. Furthermore, this growth promoting activity in culture supernatants of BL2M3 and BL312 cells was inhibited in a dose-dependent manner when cultured with mAb c143. These results suggested that TAA may be involved in the growth factor-mediated cell growth of bovine B-lymphoblastoid cell lines expressing TAA on their cell surface.     

Anti-erythrocyte membrane antibodies detected in sera of dogs naturally infected with Babesia gibsoni.

Due to the potential for anti-erythrocyte membrane antibodies as possible enhancers of erythrocyte destruction, the presence of serum anti-erythrocyte membrane antibodies in 31 dogs with Babesia gibsoni infection admitted to a veterinary hospital was investigated by an enzyme linked immunosorbent assay (ELISA) and immunoblotting analyses. This infection resulted in an increase of anti-erythrocyte membrane antibodies in 84% (IgG) and 74% (IgM) of 31 infected dogs, respectively. This was confirmed by the similarity in the protein profiles of the erythrocyte membrane antigens immunoblotted with rabbit antiserum to dog erythrocyte membrane antigens and infected dog serum. These results suggest the production of anti-erythrocyte membrane antibodies was induced by B. gibsoni infection.     

Feline Ubiquitin Fusion Protein Genes

Using cDNA from a CRFK cell line as a template, PCR amplification was performed with the Ub1S and poly(dT) primers to isolate feline ubiquitin genes. Sequencing of the 495 bp PCR fragment revealed that the putative amino acids induced by this fragment gave a fusion protein consisting of a ubiquitin polypeptide (76 amino acids) and an extension protein of ribosomal proteins L40 (52 amino acids). The putative amino acid sequence of ubiquitin was identical to those of humans, rats and pigs.The recombinant glutathione S-transferase (GST)–feline ubiquitin fusion proteins were produced in Escherichia coli and purified. The fusion proteins had a molecular weight of about 42 kDa and were detected by immunoblot assay with rabbit anti-ubiquitin antiserum.The mRNAs from heat-shocked and non-heat-shocked cells were subjected to RT-PCR (Ub1S and poly(dT) primers) analysis. The molecular weights of the ubiquitinated proteins in heat-shocked CFRK cells were between 18 kDa and 24 kDa by immunoblot assay.These results suggested that there were more ubiquinated proteins in the heat-shocked CRFK cells than in the pre-heat-shocked cells.     

Inhibitory effect of penicillin on caecal urease activity in chickens fed on a low protein diet plus urea

1. The effects of dietary penicillin on the urease activities of small intestine, large intestine, caecum and their contents, liver and kidney in chickens fed a diet containing 5 g/kg protein plus urea were examined.

2. About 0.88 of the total urease activity determined was observed in intestinal contents, of which 0.95 of the activity was accounted for by caecal contents, 0.05 by colo‐rectal contents and none by small intestinal contents. Intestinal tissues (caecum included), liver and kidney accounted for 0.03, 0.06 and 0.02, respectively, of the total urease activity.

3. Dietary penicillin decreased urease activity to 0.17 in caecal contents and to 0.05 in colo‐rectal contents of the corresponding control values (P< 0.01). The urease activity of caecal tissue was lowered by penicillin to half that of control activity (P < 0.05) but none of the activities of other tissues were affected.

4. It is concluded that, even when the urease activity is stimulated by dietary urea, 20 mg/kg dietary penicillin can strikingly lower it in the caecum, where most of the urease activity in the chicken body is to be found.     

‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3′-ends, but also the 5′-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

Effects of Latent Infection of Stock Plants and Abundance of Thrips on the Occurrence of <Emphasis Type="Italic">Tomato spotted wilt virus </Emphasis>in Chrysanthemum Fields

DAS-ELISA proved to be reliable enough to detect a latent infection by Tomato spotted wilt virus (TSWV) in asymptomatic stock plants of chrysanthemum. A high density of Frankliniella occidentalis, the predominant vector, in the presence of latently infected stock plants resulted in a high incidence of disease in the chrysanthemum production field. The incidence of disease was low when the vector thrips were not abundant in spite of the presence of latently infected stock plants. These results suggest that an infestation of the vector thrips causes severe secondary spread of TSWV originating from latently infected stock plants in chrysanthemum production fields. Received 27 July 2001/ Accepted in revised form 27 November 2001