Botulinum neurotoxins (BoNTs) are a family of protein toxins consisting of seven known serotypes (BoNT/A—BoNT/G) and multiple subtypes within the serotypes, and all of which cause the disease botulism—a disease of great public health concern. Accurate detection of BoNTs in human clinical samples is therefore an important public health goal. To achieve this goal, our laboratory developed a mass spectrometry-based assay detecting the presence of BoNT via its enzymatic activity on a peptide substrate. Recently, publications reported the use of new peptide substrates to detect BoNT/A and /B with improved results over other peptide substrates. However, the authors did not provide results of their peptide substrate on multiple subtypes of BoNT. In this work, we describe the results of testing the new substrates with multiple BoNT/A and /B subtypes and find that the substrates cannot detect many subtypes of BoNT/A and /B.

We recently identified three novel thioredoxin-like genes in the genome of the protozoan parasite Plasmodium that belong to the Phosducin-like family of proteins (PhLP). PhLPs are small cytosolic proteins hypothesized to function in G-protein signaling and protein folding. Although PhLPs are highly conserved in eukaryotes from yeast to mammals, only a few representatives have been experimentally characterized to date. In addition, while PhLPs contain a thioredoxin domain, they lack a CXXC motif, a strong indicator for redox activity, and it is unclear whether members of the PhLP family are enzymatically active. Here, we describe PbPhLP-3 as the first phosducin-like protein of a protozoan organism, Plasmodium berghei. Initial transcription analysis revealed continuous low-level expression of pbphlp-3 throughout the complex Plasmodium life cycle. Attempts to knockout pbphlp-3 in P. berghei did not yield live parasites, suggesting an essential role for the gene in Plasmodium. We cloned, expressed and purified PbPhLP-3 and determined that the recombinant protein is redox active in vitro in a thioredoxin-coupled redox assay. It also has the capacity to reduce the organic compound tert-Butyl hydroperoxide (TBHP) in vitro, albeit at low efficiency. Sequence analysis, structural modeling, and site-directed mutagenesis revealed a conserved cysteine in the thioredoxin domain to be the redox active residue. Lastly, we provide evidence that recombinant human PhLP-3 exhibits redox activity similar to that of PbPhLP-3 and suggest that redox activity may be conserved in PhLP-3 homologs of other species. Our data provide new insight into the function of PhLP-3, which is hypothesized to act as co-chaperones in the folding and regulation of cytoskeletal proteins. We discuss the potential implications of PhLP-3 as a thioredoxin-target protein and possible links between the cellular redox network and the eukaryotic protein folding machinery.

2 3 7 WEDNESDAY Supplement to Transplantation July 27, 2008, Volume 86 Number 2Sproperties. Thus, the expression of hHO-1 on porcine endothelial cells could have benefi cial effects on organ survival after xenotransplantation and could possibly interfere with XAC. In this study we analyzed porcine cells and kidneys expressing the human complement regulator CD55 and the cell protective protein hHO-1. Methods: The protective effects of the transgenes against complementmediated attack were assessed by cytotoxity assays. Organ survival and XAC following the contact of human blood with porcine endothelium, were determined by using an ex-vivo perfusion circuit based on low dose heparin mediated anticoagulation. Porcine kidneys were recovered following in situ cold perfusion with HTK organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled human AB blood. Results: Porcine fi broblast transgenic for CD55/hHO-1 showed increased resistance (up to 40%) against complement mediated attack than fi broblasts from wildtype pigs. Survival of wildtype pig kidneys during organ perfusion with human blood and addition of heparin was 42 +/-26 min. Application of soluble complement inhibition (C1-Inhibitor) prolonged organ survival to 126 +/-72 min. XAC was observed with signifi cantly elevated concentrations of d-Dimer and thrombin antithrombin complex (TAT) combined with consumption of fi brinogen and antithrombin. The histological analyses revealed numerous microthrombi. In contrast, the perfusion of the CD55/hHO-1 transgenic porcine kidneys was feasible for more than 240 min in all perfusion experiments (without C1-Inhibitor). Although elevated levels of d-Dimer and TAT were measured, no signifi cant consumption of fi brinogen and antithrombin occurred. In addition, no microthrombi were detectable histologically. Conclusion: We conclude that the expression of CD55 and hHO-1 in porcine kidneys can prolong organ survival and is able to interfere with XAC in this ex vivo perfusion model. These results suggest that the expression of hHO-1 in combination with CD55 could play an important role in xenograft protection. -defi cient (GalTKO) pig organs were developed to provide a solution to the shortage of human organ donors. These organs do not initiate hyperacute rejection but undergo acute humoral rejection mediated by xenoantibodies directed at non-gal xenoantigens. The structure, specifi city and germline origin of these antibodies has not been determined. Methods: Three rhesus monkeys were immunized with sixty million GalTKO pig endothelial cells in order to induce high levels of xenoantibodies directed at GalTKO xenoantigens. Pre-existing levels of anti-non-gal xenoantibodies were signifi cantly lower than anti-gal natural antibodies in rhesus monkeys. A strong immune response directed at GalTKO xenoantigens was confi rmed by fl ow cytometry after immunization. Pre and post transplant peripheral blood B cells were used to prepare IgG and IgM cDNA libraries for identifi cation o...