Sepsis is a common cause of death, but outcomes in individual patients are difficult to predict. Elucidating the molecular processes that differ between sepsis patients who survive and those who die may permit more appropriate treatments to be deployed. We examined the clinical features, and the plasma metabolome and proteome of patients with and without community-acquired sepsis, upon their arrival at hospital emergency departments and 24 hours later. The metabolomes and proteomes of patients at hospital admittance who would die differed markedly from those who would survive. The different profiles of proteins and metabolites clustered into fatty acid transport and β-oxidation, gluconeogenesis and the citric acid cycle. They differed consistently among several sets of patients, and diverged more as death approached. In contrast, the metabolomes and proteomes of surviving patients with mild sepsis did not differ from survivors with severe sepsis or septic shock. An algorithm derived from clinical features together with measurements of seven metabolites predicted patient survival. This algorithm may help to guide the treatment of individual patients with sepsis.

Abstract. The machinery of eukaryotic protein synthesis is found in association with the actin cytoskeleton. A major component of this translational apparatus, which is involved in the shuttling of aa-tRNA, is the actinbinding protein elongation factor let (EF-let). To investigate the consequences for translation of the interaction of EF-let with F-actin, we have studied the effect of F-actin on the ability of EF-let to bind to aa-tRNA. We demonstrate that binding of EF-let:GTP to aatRNA is not pH sensitive with a constant binding affinity of ~0.2 ~M over the physiological range of pH. However, the sharp pH dependence of binding of EF-let to F-actin is sufficient to shift the binding of EFlet from F-actin to aa-tRNA as pH increases. The ability of EF-let to bind either F-actin or aa-tRNA in competition binding experiments is also consistent with the observation that EF-let's binding to F-actin and aatRNA is mutually exclusive. Two pH-sensitive actinbinding sequences in EF-let are identified and are predicted to overlap with the aa-tRNA-binding sites. Our results suggest that pH-regulated recruitment and release of EF-let from actin filaments in vivo will supply a high local concentration of EF-let to facilitate polypeptide elongation by the F-actin-associated translational apparatus.

ABP50, an F-actin bundling protein from Dictyostelium, is also the protein synthesis co-factor, elongation factor 1 alpha (EF1 alpha). Concomitant with cAMP stimulation in Dictyostelium is a cytoplasmic alkalinization (Aerts, R. J., DeWit, R. J. W., and Van Lookeren Campagne, M. M. (1987) FEBS Lett. 220, 366-370) and a redistribution of EF1 alpha (Dharmawardhane, S., Demma, M., Yang, F., and Condeelis, J. (1991) Cell Motil. Cytoskel. 20, 279-288). In addition, others have shown a correlation between intracellular pH and the level of protein synthesis in Dictyostelium (Aerts, R. J., Durston, A. J., and Moolenaar, W. H. (1985) Cell 43, 653-657). The present study investigates the relationship between pH and the F-actin binding properties of EF1 alpha. We found that increasing pH over the physiological range 6.2-7.8 causes a loss of EF1 alpha-mediated F-actin bundling and single filament binding, with corresponding increases in the amount of free EF1 alpha in vitro. Similar results also were obtained by cell fractionation and confocal immunofluorescence microscopy. The EF1 alpha binding constant (Kd) for F-actin is increased from 0.2 microM to > 2.2 microM over the same pH range. In addition, EF1 alpha-induced actin bundle formation is freely reversible by changes in pH. Thus, pH may be a potent modulator of cytoarchitecture in Dictyostelium and may also influence mRNA translation rates by modifying the interactions between the protein synthetic machinery and the actin cytoskeleton.

Abstract. Elongation factor 1 a (EFlt~) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EFlct also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EFIot on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EFla significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EFlot nucleates actin polymerization. Although EFlct binds actin monomer, this monomer-binding activity does not explain the effects of EFlot on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EFlot. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EFla releases EFlet's inhibiting effect on actin polymerization. These data demonstrate that EFI~t can alter the assembly of F-actin, a filamentous scaffold on which nonmembrane-associated protein translation may be occurring in vivo.

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