The cytoplasmic heritable determinant [PSI ؉ ] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSI ؉ ] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [ curing ͉ Sup35p ͉ cytoplasmic determinant R ecent biochemical evidence (1-5) has supported the hypothesis that the [PSI ϩ ] phenotype of Saccharomyces cerevisiae reflects the prion-like properties of the SUP35 gene product (6, 7). The essential chromosome-encoded protein Sup35p is known to be one of two eukaryote polypeptide release factors, namely eRF3 (8, 9). Sup35p associates with Sup45p (eRF1) in vivo to mediate translation termination (8). In vitro, Sup35p forms highly ordered fibers, whose appearance resembles that of fibrils formed by other amyloidogenic polypeptides (3,4 , a second prion determinant of S. cerevisiae (7). Other reagents including methanol, ethylene glycol, and hypertonic conditions, have been reported to exhibit curing properties; however, none of these cure with the neartotal efficiency of GuHCl (13,14).Two hypotheses have been proposed to account for the curing properties of GuHCl. First, the elimination of the prion might arise directly from the ability of GuHCl to denature proteins. However, the concentrations of GuHCl effective in curing [PSI ϩ ] are in the millimolar range, rather than the molar range typically required for the denaturation of proteins in vitro (13). Alternatively, GuHCl may actually promote the expression of an ancillary factor, namely the stress protein Hsp104p, which indirectly results in the reactivation of Sup35p and consequently the loss of the prion (15). Unlike other heat shock proteins, Hsp104p does not act to protect proteins against stress (i.e., heat denaturation), rather Hsp104p actively promotes the recovery of stress-denatured aggregated proteins by facilitating their refolding back into functional, native conformations (16,17). Overexpression of Hsp104p might lead to the total refolding of Sup35p from the aberrant prion conformation to its native structure, thereby mediating prion loss. To test both hypotheses, we have examined the kinetics of prion elimination upon growth in the presence of GuHCl and assessed the influence of stress on the curing process. Materials and MethodsStrain. The genotype of the strain used in this study was BSC783͞4a: , MATa. Growth Media. BSC783͞4a was grown at 30°C on 1 ⁄4YEPD solid medium [4% (wt͞vol) glucose, 1% (wt͞vol) Bacto-peptone, 0.25% (wt͞vol) yeast extract, 2% (wt͞vol) agar]. Most liquid cultures also were grown at 30°C in YEPD complete medium [2% (wt͞vol) glucose, 1% (wt͞vol) Bacto-peptone, 1% (wt͞vol) yeast extract], with or without 3 mM GuHCl. For studies using ethanol-supplemented media, strains were grown in f...

Purpose: Contemporary trauma resuscitation prioritizes control of bleeding and uses major haemorrhage protocols (MHPs) to prevent and treat coagulopathy. We aimed to determine whether augmenting MHPs with Viscoelastic Haemostatic Assays (VHA) would improve outcomes compared to Conventional Coagulation Tests (CCTs). Methods: This was a multi-centre, randomized controlled trial comparing outcomes in trauma patients who received empiric MHPs, augmented by either VHA or CCT-guided interventions. Primary outcome was the proportion of subjects who, at 24 h after injury, were alive and free of massive transfusion (10 or more red cell transfusions). Secondary outcomes included 28-day mortality. Pre-specified subgroups included patients with severe traumatic brain injury (TBI). Results: Of 396 patients in the intention to treat analysis, 201 were allocated to VHA and 195 to CCT-guided therapy. At 24 h, there was no difference in the proportion of patients who were alive and free of massive transfusion (VHA: 67%, CCT: 64%, OR 1.15, 95% CI 0.76-1.73). 28-day mortality was not different overall (VHA: 25%, CCT: 28%, OR 0.84, 95% CI 0.54-1.31), nor were there differences in other secondary outcomes or serious adverse events. In pre-specified subgroups, there were no differences in primary outcomes. In the pre-specified subgroup of 74 patients with TBI, 64% were alive and free of massive transfusion at 24 h compared to 46% in the CCT arm (OR 2.12, 95% CI 0.84-5.34). Conclusion: There was no difference in overall outcomes between VHA-and CCT-augmented-major haemorrhage protocols.

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The importance of receptor binding in the potent immunogenicity of Escherichia coli heat-labile enterotoxin B subunit (EtxB) was tested by comparing its immunogical properties with those of a receptor binding mutant, EtxB(G33D). Subcutaneous immunization of EtxB(G33D) resulted in 160-fold reduction in antibody titer compared with wild-type EtxB, whereas its oral delivery failed to provoke any detectable secretory or serum anti-B subunit responses. Moreover, the two proteins induced strikingly different effects on lymphocyte cultures in vitro. EtxB, in comparison with EtxB(G33D), caused an increase in the proportion of B cells, many ofwhich were activated (CD25+); the complete depletion of CD8+ T cells; an increase in the activation of CD4+ T cells; and an increase in interleukin 2 and a decrease in interferon 'y. These data indicate that EtxB exerts profound effects on immune cells, suggesting that its potent immunogenicity is dependent not only on efficient receptor-mediated uptake, but also on direct receptor-mediated immunomodulation of lymphocyte subsets.

We describe a systematic approach to define threshold parameters for ROTEM and TEG. These parameters were incorporated into algorithms to support data-driven adjustments of resuscitation with therapeutics, to optimize damage control resuscitation practice in trauma.