Metabolites in Blood for Prediction of Bacteremic Sepsis in the Emergency Room

A metabolomics approach for prediction of bacteremic sepsis in patients in the emergency room (ER) was investigated. In a prospective study, whole blood samples from 65 patients with bacteremic sepsis and 49 ER controls were compared. The blood samples were analyzed using gas chromatography coupled to time-of-flight mass spectrometry. Multivariate and logistic regression modeling using metabolites identified by chromatography or using conventional laboratory parameters and clinical scores of infection were employed. A predictive model of bacteremic sepsis with 107 metabolites was developed and validated. The number of metabolites was reduced stepwise until identifying a set of 6 predictive metabolites. A 6-metabolite predictive logistic regression model showed a sensitivity of 0.91(95% CI 0.69–0.99) and a specificity 0.84 (95% CI 0.58–0.94) with an AUC of 0.93 (95% CI 0.89–1.01). Myristic acid was the single most predictive metabolite, with a sensitivity of 1.00 (95% CI 0.85–1.00) and specificity of 0.95 (95% CI 0.74–0.99), and performed better than various combinations of conventional laboratory and clinical parameters. We found that a metabolomics approach for analysis of acute blood samples was useful for identification of patients with bacteremic sepsis. Metabolomics should be further evaluated as a new tool for infection diagnostics.     

Comparative study of the microtriches of adult Cestodes (Proteocephalidea: Monticelliidae), and some comments on their systematic value

The surface of the tegument of the scolex and the immature proglottides of Monticellia belavistensis, M. ventrei, Nomimoscolex chubbi, and N. lopesi is described using scanning electron microscopy. Only blade-like spiniform microtriches and filiform microtriches were observed in the species studied. The types, size and density of microtriches on the apical region surface of the scolex, central cavity surface of suckers, marginal ring surface of suckers, non-adherent surface of suckers, proliferation zone surface, and immature proglottis surface were compared among these species. The distribution pattern of the microtriches was not a reliable feature to discriminate among the genera considered in this study. It varied in each of the species of Monticellia examined, and did not permit to split the heterogeneous genus Nomimoscolex. However, the microthrix pattern can be regarded as an additional diagnostic feature to distinguish among species of proteocephalideans. Further comparative research involving other species of proteocephalid taxa is needed to elucidate the systematic value of the tegumental morphology.     

What Can We Learn from the Evolution of Protein-Ligand Interactions to Aid the Design of New Therapeutics?

Efforts to increase affinity in the design of new therapeutic molecules have tended to lead to greater lipophilicity, a factor that is generally agreed to be contributing to the low success rate of new drug candidates. Our aim is to provide a structural perspective to the study of lipophilic efficiency and to compare molecular interactions created over evolutionary time with those designed by humans. We show that natural complexes typically engage in more polar contacts than synthetic molecules bound to proteins. The synthetic molecules also have a higher proportion of unmatched heteroatoms at the interface than the natural sets. These observations suggest that there are lessons to be learnt from Nature, which could help us to improve the characteristics of man-made molecules. In particular, it is possible to increase the density of polar contacts without increasing lipophilicity and this is best achieved early in discovery while molecules remain relatively small.     

N-Acetylsuccinimidylglutamate, a Cyclic Imide Form of the Peptide N-Acetylaspartylglutamate, Is Present in Low Micromolar Concentrations in Murine and Bovine CNS

Abstract: N -Acetylsuccinimidylglutamate [(asu)NAAG], a cyclic form of the peptide N -acetylaspartylglutamate (NAAG) in which the aspartyl residue is linked to glutamate via the α- and β-carboxylates, was identified and quantified by HPLC in the murine and bovine CNS. In the rat, the highest concentrations of (asu)NAAG were detected in the spinal cord (1.83 ± 0.15 pmol/mg of wet tissue weight) and brainstem (1.16 ± 0.08 pmol/mg wet weight), whereas the levels were below the limit of detection in cerebellum, hippocampus, and cerebral cortex. (Asu)NAAG was also detected in significant amounts in the superior colliculus and lateral genicutale nucleus (1.17 ± 0.05 and 0.82 ± 0.13 pmol/mg wet weight, respectively). Although the tissue content of (asu)NAAG was about three orders of magnitude lower than that of NAAG, levels of both peptides were positively correlated among different CNS regions ( r = 0.74, p < 0.003). In the rat spinal cord, (asu)NAAG levels progressively increased from week 2 to month 12 after birth. In bovine spinal cord, the contents of (asu)NAAG and NAAG were comparable in gray and white matter as well as in the dorsal and ventral horns. These results suggest that NAAG and (asu)-NAAG are closely related metabolically and raise the question of the physiological significance of such a cyclic peptide.     

Inactivation of mitochondrial carbamoyl phosphate synthetase induced by ascorbate, oxygen, and Fe3+ in the presence of acetylglutamate: protection by ATP and HCO3- and lack of inactivation of ornithine transcarbamylase

Of the two mitochondrial enzymes of the urea cycle, carbamoyl phosphate synthetase (CPS) was and ornithine transcarbamylase (OTC) was not inactivated by the Fe3+-oxygen-ascorbate model system for mixed-function oxidation [R. L. Levine, (1983) J. Biol. Chem. 258, 11828-11833]. The susceptibility of OTC was not increased by its substrates, products, or inhibitors, whereas that of CPS was markedly increased by acetylglutamate (its allosteric activator) when ATP was absent. Thus, acetylglutamate binds in the absence of ATP and exposes to oxidation essential groups of the enzyme. We estimate for this binding a KD value of 1.6 mM, which greatly exceeds the KD values (less than 10 microM) determined in the presence of ATP and bicarbonate. ATP, and even more, mixtures of ATP and bicarbonate protected CPS from inactivation. Acetylglutamate exposes the site for the ATP molecule that yields Pi, and it appears that ATP protects by binding at this site. Experiments of limited proteolysis with elastase suggest that oxidation prevents this binding of ATP and show that it accelerates cleavage of CPS by the protease, thus supporting the idea that oxidation may precede proteolysis. Trypsin, chymotrypsin, and papain also hydrolyze the oxidized enzyme considerably faster than the native enzyme. Our results also support the idea that oxidative inactivation is site specific and requires sites on the enzyme for Me2+ and, possibly, for a nucleotide.     

Limited proteolysis reveals low-affinity binding of N-acetyl-L-glutamate to rat-liver carbamoyl-phosphate synthetase (ammonia)

Carbamoyl-phosphate synthetase was inactivated by elastase with first-order kinetics, and N-acetyl-L-glutamate speeded inactivation. From the dependence of the t1/2 value for inactivation on the concentration of acetylglutamate we estimate a Kd value for binding of the activator of 0.365 mM, which is approximately 600 times greater than in the presence of ATP, HCO3-, K+ and Mg2+. K+ and Mg2+ are not required for binding with low affinity, and in the absence of ATP they do not appear to increase the affinity for acetylglutamate. In the presence of acetylglutamate, mixtures of ATP, K+ and Mg2+ protect the enzyme from inactivation. ADP or AdoPP[NH]P partly replaced ATP in protecting the enzyme and thus binding of the nucleotide without further reaction is enough for protection. Two partial activities of the enzyme were inactivated by elastase to the same extent as the overall reaction, and thus elastase affects some property of the enzyme which is essential for catalysis. With other proteinases tested, inactivation was also accelerated by acetylglutamate and was slowed by mixtures of ATP, K+, Mg2+ and acetylglutamate, suggesting that changes in the accessibility of susceptible bonds are responsible for the changes in the degree of inactivation. It is concluded that elastase attacks at or close to the binding sites for ATP, and that exposure of the binding site for the ATP molecule that yields Pi (ATPA) upon binding of acetylglutamate causes the acceleration of the proteolytic inactivation.