There is considerable interest in the potential of Epstein-Barr virus (EBV) latent antigen-specific CD4؉ T cells to act as direct effectors controlling EBV-induced B lymphoproliferations. Such activity would require direct CD4؉ T-cell recognition of latently infected cells through epitopes derived from endogenously expressed viral proteins and presented on the target cell surface in association with HLA class II molecules. It is therefore important to know how often these conditions are met. Here we provide CD4؉ epitope maps for four EBV nuclear antigens, EBNA1, -2, -3A, and -3C, and establish CD4 ؉ T-cell clones against 12 representative epitopes. For each epitope we identify the relevant HLA class II restricting allele and determine the efficiency with which epitope-specific effectors recognize the autologous EBV-transformed B-lymphoblastoid cell line (LCL). The level of recognition measured by gamma interferon release was consistent among clones to the same epitope but varied between epitopes, with values ranging from 0 to 35% of the maximum seen against the epitope peptide-loaded LCL. These epitope-specific differences, also apparent in short-term cytotoxicity and longer-term outgrowth assays on LCL targets, did not relate to the identity of the source antigen and could not be explained by the different functional avidities of the CD4 ؉ clones; rather, they appeared to reflect different levels of epitope display at the LCL surface. Thus, while CD4 ؉ T-cell responses are detectable against many epitopes in EBV latent proteins, only a minority of these responses are likely to have therapeutic potential as effectors directly recognizing latently infected target cells.
The anticancer activity of disulfiram (DS) is copper(ii) (Cu)-dependent.
Sulfation of the amino acid residues of proteins is a significant post‐translational modification, the functions of which are yet to be fully understood. Current sulfation methods are limited mainly to O‐tyrosine (sY), which requires negatively charged species around the desired amino acid residue and a specific sulfotransferase enzyme. Alternatively, for solid‐phase peptide synthesis, a de novo protected sY is required. Therefore, synthetic routes that go beyond O‐sulfation are required. We have developed a novel route to N‐sulfamation and can dial‐in/out O‐sulfation (without S‐sulfurothiolation), mimicking the initiation step of the ping‐pong sulfation mechanism identified in structural biology. This rapid, low‐temperature and non‐racemising method is applicable to a range of amines, amides, amino acids, and peptide sequences.
The first synthesis of kottamide E, a marine natural product containing a 5,6-dibromoindole linked via a (Z)-enamide to an unusual 1,2-dithiolane-containing amino acid, is reported.
In the present work we develop a real-time electrochemical mediator assay to enable the assessment of cell numbers and chemical toxicity. This allowed us to monitor metabolism down to a single cell in a low cost easy to use rapid assay which is not possible with current technology. The developed assay was based on the determination of oxygen. This was made possible via the use of electrochemical mediator ferrocene carboxylic acid (FcA). The FcA showed distinctive catalytic properties in interacting with reactive oxygen species generated from oxygen when compared to ferrocene methanol (FcMeOH). A deeper insight into the chemistry controlling this behaviour is provided. The behaviour is then taken advantage of to develop a cellular aerobic respiration assay. We describe the properties of the FcA system to detect, in real-time, the oxygen consumption of Escherichia coli DH5-α (E. coli). We demonstrated that the FcA-based oxygen assay is highly sensitive, and using a population of cells, oxygen consumption rates could be calculated down to a single cell level. More importantly, the results can be accomplished in minutes, considerably outperforming current commercially available biooxygen demand assays. The developed assay is expected to have a significant impact in diverse fields and industries, ranging from environmental toxicology through to pharmaceutical and agrochemical industries.
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