Recombinant exoenzyme S (rHisExoS) of Pseudomonas aeruginosa was expressed in Escherichia coli as a soluble, cytosolic His fusion protein. rHisExoS was purified by Ni 2؉-affinity chromatography in the presence of protease inhibitors without detectable degradation. rHisExoS possessed a specific activity (within twofold) for the factor-activating exoenzyme S-dependent ADP-ribosylation of soybean trypsin inhibitor (SBTI) similar to that of native exoenzyme S. Analysis of several deletion peptides showed that ⌬N222, which encoded the carboxyl-terminal 222 amino acids of exoenzyme S, possessed factor-activating exoenzyme S-dependent ADPribosyltransferase activity. ⌬N222 catalyzed the ADP-ribosylation of SBTI at a rate sixfold greater than rHisExoS. Relative to rHisExoS, ⌬N222 had a similar affinity for NAD, a threefold greater affinity for SBTI, and a four-to eightfold greater k cat for the ADP-ribosylation of SBTI. Like native exoenzyme S, rHisExoS chromatographed as an aggregate with an apparent molecular mass of >300 kDa. In contrast, ⌬N222 did not chromatograph as an aggregate, which showed that the amino-terminal 99 amino acids of exoenzyme S were responsible for the aggregation phenotype.

This qualitative study is concerned with the views of clergy about the constituent elements of a 'good death'. The method employed to explore the question was a set of cards called the 'Conversations Game'. These cards, developed from research in the USA in 1999, contain a list of identified ingredients of a 'good death'. Within this study, the cards were further ordered to conceptualise the field under four headings, covering the four elements of palliative care: physical care, emotional (or psychological) care, social care, and spiritual care. Choices made from the cards formed the content of structured interviews conducted in November and December 2011 with a randomly selected group of Church of England clergy from the Diocese of Worcester. The study indicated that the clergy interviewed differed in their chosen components of a good death from the professional consensus in healthcare in some respects. Chief among these was a high priority given to spiritual care concerns.

Pseudomonas aeruginosa produces two ADP-ribosyltransferases, exotoxin A and exoenzyme S (ExoS). Although the physiological target protein remains to be defined, ExoS has been shown to ADP-ribosylate several eukaryotic proteins in vitro, including vimentin and members of the family of low-molecular-weight GTPbinding proteins. Recently, ExoS ADP-ribosyltransferase activity has been detected in the pleural fluid of rabbits infected with P. aeruginosa. This observation prompted an examination of the potential for ExoS to function as an ecto-ADP-ribosyltransferase. We have observed that ExoS preferentially ADP-ribosylated two extracellular serum proteins with molecular masses of 150 and 27 kDa. The ADP-ribosylation of these serum proteins by ExoS was stimulated by, but not dependent upon, exogenous FAS (for factor activating exoenzyme S), which indicated that serum contained endogenous FAS activity. Biochemical analysis showed that the 150-kDa ADP-ribosylated protein was immunoglobulin of the immunoglobulin G (IgG) and IgA classes. Subtyping showed that ExoS preferentially ADP-ribosylated human IgG3 and that ADP-ribosylation occurred within its Fc region. The 27-kDa protein ADP-ribosylated by ExoS was determined to be apolipoprotein A1. These data demonstrate ecto-ADP-ribosyltransferase activity by ExoS. This may extend the potential physiological consequences of ExoS during infection by P. aeruginosa beyond the implicated type III secretionmediated intracellular delivery of ExoS into sensitive eukaryotic cells.

Our results suggest that the PSMA peptide LLHETDSAV is poorly immunogenic in humans and that alternative prostate cancer antigens should be pursued.

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