Soluble C3 proconvertase and convertase of the classical pathway of human complement. Conditions of stabilization in vitro.

Soluble classical-pathway C3 convertase and proconvertase were prepared from purified C4b-C2ox complex in the presence of Ni2+; the two complexes, stable for at least 15 h at 4 degrees C, were isolated by sucrose-density-gradient ultracentrifugation. The C3 convertase alone was able to cleave C3, and its decay was accelerated in the presence of C4-binding protein. The individual roles of Ni2+ and I2 treatment of C2 in the stabilization of the complexes seemed to be different and additive. 63Ni2+ binding coupled to h.p.l.c. analysis showed that 63Ni2+ bound only to the C2ox proteolytic fragment a (1 mol/mol) with a Kd of 26 microM. Competition studies between Ni2+ and Mg2+ indicated that only half of the Ni2+ bound to the C3 convertase was removed by Mg2+, whereas, in the same conditions, Ni2+ bound to C2ox proteolytic fragment a was not displaced, suggesting the presence of two sets of sites on the convertase. EDTA prevented the formation of both C3 convertase and proconvertase; EDTA had no effect on the preformed C3 convertase, whereas it dissociated the preformed proconvertase.     

Detoxification of naturally occurring chromenes in larvae of the generalist herbivore Spodoptera littoralis (Noctuidae)

Five naturally occurring chromenes from the Asteraceae including the insecticidal compounds precocene II (1) and encecalin (2) were administered to last instar larvae of Spodoptera littoralis via the food or by topical contact. Metabolites formed and excreted via the frass were analysed by GC-MS and by direct comparison with reference compounds obtained by partial synthesis. In total 28 different metabolites were identified, many of them reported here for the first time. All metabolites detected originated from phase I reactions (most probably catalysed by Cytochrom P-450-dependent monooxygenases) by hydroxylation, demethylation or reduction of the parent chromenes. The resulting metabolites can be regarded as detoxification products based on previous structure-activity studies. The increased polarity of the metabolites will furthermore facilitate their excretion by the larvae compared to the more apolar parent chromenes. The largest number of metabolites (eight for each compound) was detected following oral treatment with precocene II and encecalin respectively. 3-Monool as well as 3,4-trans-diol derivatives predominated in the frass of larvae treated with the latter compounds whereas the 6-hydroxyethyl derivatives were the major metabolites of the other chromenes investigated. The patterns of metabolites originating from precocene II or encecalin were the same following oral application or topical treatment.     

Postoperative peak serum C-reactive protein is a predictor of outcome following liver transplantation for hepatocellular carcinoma

Context: C-reactive protein (CRP), a biomarker of inflammation, may correlate with prognosis in several malignancies.

Objective: To investigate the prognostic impact of early postoperative peak serum levels of CRP on tumor-specific outcome in 106 liver transplant patients with hepatocellular carcinoma (HCC).

Methods and results: In multivariate Cox regression analysis, a posttransplant elevated peak CRP level (>versus?≤?3.5?mg/dl) was identified as an independent predictor of poor recurrence-free survival (p?=?0.01; HR?=?4.04; CI?=?1.399–11.640).

Conclusion: Early postoperative serum CRP may serve as a useful inflammation-based biomarker of outcome in liver transplant patients with HCC.     

Antigen presentation in Syrian hamster cells: substrate selectivity of TAP controlled by polymorphic residues in TAP1 and differential requirements for loading of H2 class I molecules

Expression of mouse major histocompatibility complex (MHC) class I molecules in different cell lines derived from Syrian hamsters has revealed antigen presentation deficiencies of some H2 allelic products in two cell lines (BHK and NIL-2) which were overcome by transient expression of the rat transporter associated with antigen processing (TAP; Lobigs et al. 1995). Here we show that in both cell lines the endogenous MHC class I cell surface expression was completely down-regulated. Lymphokine treatment induced endogenous and recombinant mouse MHC class I cell surface expression to levels similar to that in other Syrian hamster cell lines competent for antigen presentation through transduced H2 molecules. Accordingly, constitutive downregulation of expression of accessory molecules of the MHC class I pathway can reveal differences between H2 class I alleles in antigen presentation not encountered when the expression levels are augmented. In addition to the differential expression of MHC class I pathway genes, two cell lines representing competent (FF) and defective (BHK) antigen presentation phenotypes for mouse class I MHC restriction elements demonstrated substantial sequence polymorphism in Tap1 but not Tap2. Cytokine-treated FF or BHK cells and human TAP-deficient T2 cells transfected with FF or BHK TAP1 in combination with FF TAP2 differed in their preference for C-terminal peptide residues, as shown by an in vitro peptide transport assay. Thus, polymorphic residues in TAP1 can influence the substrate selectivity of the Syrian hamster peptide transporter.     

The ubiquitin ligation machinery in the defense against bacterial pathogens

The ubiquitin system is an important part of the host cellular defense program during bacterial infection. This is in particular evident for a number of bacteria including Salmonella Typhimurium and Mycobacterium tuberculosis which—inventively as part of their invasion strategy or accidentally upon rupture of seized host endomembranes—become exposed to the host cytosol. Ubiquitylation is involved in the detection and clearance of these bacteria as well as in the activation of innate immune and inflammatory signaling. Remarkably, all these defense responses seem to emanate from a dense layer of ubiquitin which coats the invading pathogens. In this review, we focus on the diverse group of host cell E3 ubiquitin ligases that help to tailor this ubiquitin coat. In particular, we address how the divergent ubiquitin conjugation mechanisms of these ligases contribute to the complexity of the anti‐bacterial coating and the recruitment of different ubiquitin‐binding effectors. We also discuss the activation and coordination of the different E3 ligases and which strategies bacteria evolved to evade the activities of the host ubiquitin system.