No abstract

Protein-protein interactions play a critical role in cellular processes such as signal transduction. Although many methods for identifying the binding partners of a protein of interest are available, it is currently difficult or impossible to assess the functional consequences of a specific interaction in vivo. To address this issue, we propose to modify proteins by addition of an artificial protein binding interface, thereby forcing them to interact in the cell in a pairwise fashion and allowing the functional consequences to be determined. For this purpose, we have developed an artificial binding interface consisting of a anti-Myc single-chain antibody (ScFv) and its peptide epitope. We found that the binding of an ScFv derived from anti-Myc monoclonal antibody 9E10 was relatively weak in vivo, so we selected an improved clone, 3DX, by in vitro mutagenesis and phage display. 3DX bound well to its epitope in a yeast two-hybrid system, and GST-fused 3DX also bound to several Myc-tagged proteins in mammalian cells. In vivo binding was relatively insensitive to the position of the ScFv in a fusion protein, but was improved by including multiple tandem copies of the Myc epitope in the binding partner. To test the system, we successfully replaced the SH3 domain-mediated interaction between the Abl tyrosine kinase and adaptor proteins Crk and Nck with an engineered interaction between 3DX and multiple Myc tags. We expect that this approach, which we term a functional interaction trap, will be a powerful proteomic tool for investigating protein-protein interactions.

Nickel is the major cause of metal-induced allergic dermatitis. Twelve nickel-specific T cell clones were used to investigate the cellular immune reactions occurring in nickel sensitivity. The selection between the alternative T cell receptors alpha beta and gamma delta and two alternative V beta genes (V beta 5 and V beta 8) were studied to see if nickel induces a selective pressure for clones bearing particular genes. Cell surface markers were studied by monoclonal antibodies and flow cytometry. Soluble mediators were measured by an ELISA method. The clones used T cell receptor alpha beta genes but did not use V beta 5 or V beta 8. They were T helper clones with a primed memory marker (CD3+ CD4+ CD8- CD45RO+) and carried HLA-DR. None of the clones secreted IL-1 alpha, all of them secreted IL-2 receptor. Four clones secreted IL-1 beta, six IL-4 and seven IL-6, the peaks in IL-2R and IL-6 secretion preceding IL-4 secretion. The clones helped immunoglobulin synthesis. The clones from late effector phase of the nickel allergic reaction favours the use of T cell receptors alpha beta genes. Nickel-specific clones were phenotypically indistinguishable but differed in soluble mediators produced.

– Salivary immunoglobulins, lysozyme, pH, and microbial counts were determined in 55 children with cancer diseases (37 cured subjects and 18 acute ones) and 103 healthy subjects. 5–10 ml unstimulated whole saliva was collected and pH, immunoglobulins and lysozyme were measured. Chairside dip‐slide cultivations were used for microbiologic cultures. Reduced salivary pH and an increased amount of lysozyme were found in the saliva of those children who had been cured of their cancer diseases, but ongoing cancer disease or the treatment provided for it reduced pH and increased the amounts of lysozyme, lactobacillus, Streptococcus mutans and some immunoglobulins. These findings suggest that children with childhood cancer may be more susceptible to dental diseases than healthy ones.

SUMMARYHelicobacter pylori-induced in vitro stimulation of mononuclear cells was characterized by measuring DNA synthesis response, interferon-gamma (IFN-y) secretion and the number of immunoglobulinsecreting cells. The strength of these responses was measured in 51 subjects comprising 36 dyspeptic patients from the Gastroenterological Unit and 15 members ofthe laboratory staff. Nineteen subjects had antibodies to H. pylori and 32 did not. The responses were compared with respect to H. pylori antibody status. Positive stimulation of DNA synthesis (stimulation index > 2) was obtained in 96% of the subjects, and the bacterium induced IFN-y secretion in all of them. The induction of immunoglobulin-secreting cells revealed B cell in vitro stimulation. The antibody-positive subjects had a tendency to synthesize less DNA (Pnol significant) and secrete less I FN-y (F < 0-05) in response to H. pylori than did the antibody-negative ones, but the differences were not marked. The results show that the whole H. pylori bacterium stimulates mononuclear cells. The nature ofthe stimulation is not yet characterized (non-specific versus specific).