TNFα-Mediated Liver Destruction by Kupffer Cells and Ly6Chi Monocytes during Entamoeba histolytica Infection

Amebic liver abscess (ALA) is a focal destruction of liver tissue due to infection by the protozoan parasite Entamoeba histolytica (E. histolytica). Host tissue damage is attributed mainly to parasite pathogenicity factors, but massive early accumulation of mononuclear cells, including neutrophils, inflammatory monocytes and macrophages, at the site of infection raises the question of whether these cells also contribute to tissue damage. Using highly selective depletion strategies and cell-specific knockout mice, the relative contribution of innate immune cell populations to liver destruction during amebic infection was investigated. Neutrophils were not required for amebic infection nor did they appear to be substantially involved in tissue damage. In contrast, Kupffer cells and inflammatory monocytes contributed substantially to liver destruction during ALA, and tissue damage was mediated primarily by TNFα. These data indicate that besides direct antiparasitic drugs, modulating innate immune responses may potentially be beneficial in limiting ALA pathogenesis.     

The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NFκB Transcription Factors

Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells.     

Isoflavone synthase (IFS) gene phylogeny in Trifolium species associated with plant isoflavone contents

This study presents the results of the identification and quantification of 12 isoflavones (prunetin, irilone, pseudobaptigenin, glycitein, daidzin, genistin, daidzein, pratensein, puerarin, biochanin A, formononetin and genistein) in 23 species of Trifolium (T. arvense, T. pratense, T. ligusticum, T. striatum, T. lappaceum, T. angustifolium, T. hirtum, T. subterraneum, T. isthmocarpum, T. stellatum, T. mutabile, T. strictum, T. fragiferum, T. alexandrinum, T. tomentosum. T. nigrescens subsp. petrisavii, T. nigrescens, T. glomeratum, T. subterraneum subsp. brachycalycinum, T. cherleri, T. resupinatum, T. campestre and T. repens). Isoflavones were extracted by an MSPD method and analyzed with HPLC coupled with a diode-array detector. The evaluation of molecular phylogeny of the IFS gene and the relation with isoflavone content was also performed. Five species (T. subterraneum subsp. brachycalycinum, T. alexandrinum, T. pratense, T. subterraneum and T. lappaceum) were identified with high levels of biochanin A (431–83 mg/kg), formononetin (72–365 mg/kg) and genistein (9–509 mg/kg), which could be utilized as alternative sources for the nutraceutical industry. Genetic phylogeny for the IFS gene was found in the species studied, with 20 out of 23 species having been divided into two clades, while the remaining three were genetically distant. Based on our results, we confirm the direct correlation between IFS gene polymorphism and isoflavones content in species of Trifolium particularly noted for formononetin. Therefore, the IFS gene can be utilized for screening Trifolium genotypes for formononetin. The relation of the three isoflavones' contents and the molecular phylogeny of plants determined by the IFS sequences, as a screening marker for plants with high isoflavone contents in Trifolium species, are to the best of our knowledge described for the first time.     

Disorder and order in unfolded and disordered peptides and proteins: A view derived from tripeptide conformational analysis. II. Tripeptides with short side chains populating asx and β‐type like turn conformations

In the preceding paper, we found that ensembles of tripeptides with long or bulky chains can include up to 20% of various turns. Here, we determine the structural and thermodynamic characteristics of GxG peptides with short polar and/or ionizable central residues (D, N, C), whose conformational distributions exhibit higher than average percentage (>20%) of turn conformations. To probe the side‐chain conformations of these peptides, we determined the ³J(H^α,H^β) coupling constants and derived the population of three rotamers with χ₁‐angles of ?60°, 180° and 60°, which were correlated with residue propensities by DFT‐calculations. For protonated GDG, the rotamer distribution provides additional evidence for asx‐turns. A comparison of vibrational spectra and NMR coupling constants of protonated GDG, ionized GDG, and the protonated aspartic acid dipeptide revealed that side chain protonation increases the pPII content at the expense of turn populations. The charged terminal groups, however, have negligible influence on the conformational properties of the central residue. Like protonated GDG, cationic GCG samples asx‐turns to a significant extent. The temperature dependence of the UVCD spectra and ³J(H^NH^α) constants suggest that the turn populations of GDG and GNG are practically temperature‐independent, indicating enthalpic and entropic stabilization. The temperature‐independent J‐coupling and UVCD spectra of GNG require a three‐state model. Our results indicate that short side chains with hydrogen bonding capability in GxG segments of proteins may serve as hinge regions for establishing compact structures of unfolded proteins and peptides. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

A predictive framework to assess spatio‐temporal variability of infestations by the European spruce bark beetle

Natural disturbances are key factors for the development of forest ecosystems. In forests of central Europe and Scandinavia, the European spruce bark beetle Ips typographus is the most devastating biotic disturbance agent in Norway spruce Picea abies, but our understanding of the factors determining its spatio‐temporal dynamics is still quite limited. To quantify the drivers of bark beetle dynamics, we analyzed a survey dataset with annual resolution that covers 17 yr and 469 forest districts (10 860 km² of forest area) all over Switzerland. We used Poisson log‐normal models in a Bayesian framework to analyze the spatio‐temporal dynamics of bark beetle infestation spots at the forest district level. Bark beetle infestations increased with increasing heat sum (> 8.3°C), volume of standing Norway spruce stock, and the number of infestation spots of the previous year. Precipitation tended to slightly affect the risk of bark beetle infestations. Two major storm events further increased the spatio‐temporal variability of bark beetle infestations. Spruce abundance, storm damage and temperature are known to be important factors influencing the population dynamics of the European spruce bark beetle. Our study is the first to quantify the combined effects of spruce abundance and heat sum, whereby the heat sum turned out to be the most important and consistent predictor. Because our study area encompasses large ecological and climatological gradients, our model is likely to be applicable to Norway spruce forests in other regions of central Europe and Scandinavia.     

Switching the Clientele: A LYSINE RESIDING IN THE C TERMINUS OF THE SEROTONIN TRANSPORTER SPECIFIES ITS PREFERENCE FOR THE COAT PROTEIN COMPLEX II COMPONENT SEC24C*

The serotonin transporter (SERT) maintains serotonergic neurotransmission via rapid reuptake of serotonin from the synaptic cleft. SERT relies exclusively on the coat protein complex II component SEC24C for endoplasmic reticulum (ER) export. The closely related transporters for noradrenaline and dopamine depend on SEC24D. Here, we show that discrimination between SEC24C and SEC24D is specified by the residue at position +2 downstream from the ER export motif (⁶⁰⁷RI⁶⁰⁸ in SERT). Substituting Lys⁶¹⁰ with tyrosine, the corresponding residue found in the noradrenaline and dopamine transporters, switched the SEC24 isoform preference: SERT-K610Y relied solely on SEC24D to reach the cell surface. This analysis was extended to other SLC6 (solute carrier 6) transporter family members: siRNA-dependent depletion of SEC24C, but not of SEC24D, reduced surface levels of the glycine transporter-1a, the betaine/GABA transporter and the GABA transporter-4. Experiments with dominant negative versions of SEC24C and SEC24D recapitulated these findings. We also verified that the presence of two ER export motifs (in concatemers of SERT and GABA transporter-1) supported recruitment of both SEC24C and SEC24D. To the best of our knowledge, this is the first report to document a change in SEC24 specificity by mutation of a single residue in the client protein. Our observations allowed for deducing a rule for SLC6 family members: a hydrophobic residue (Tyr or Val) in the +2 position specifies interaction with SEC24D, and a hydrophilic residue (Lys, Asn, or Gln) recruits SEC24C. Variations in SEC24C are linked to neuropsychiatric disorders. The present findings provide a mechanistic explanation. Variations in SEC24C may translate into distinct surface levels of neurotransmitter transporters.