Structural basis for innate immune sensing by M-ficolin and its control by a pH-dependent conformational switch

Ficolins are soluble oligomeric proteins with lectin-like activity, assembled from collagen fibers prolonged by fibrinogen-like recognition domains. They act as innate immune sensors by recognizing conserved molecular markers exposed on microbial surfaces and thereby triggering effector mechanisms such as enhanced phagocytosis and inflammation. In humans, L- and H-ficolins have been characterized in plasma, whereas a third species, M-ficolin, is secreted by monocytes and macrophages. To decipher the molecular mechanisms underlying their recognition properties, we previously solved the structures of the recognition domains of L- and H-ficolins, in complex with various model ligands (Garlatti, V., Belloy, N., Martin, L., Lacroix, M., Matsushita, M., Endo, Y., Fujita, T., Fontecilla-Camps, J. C., Arlaud, G. J., Thielens, N. M., and Gaboriaud, C. (2007) EMBO J. 24, 623-633). We now report the ligand-bound crystal structures of the recognition domain of M-ficolin, determined at high resolution (1.75-1.8 A), which provides the first structural insights into its binding properties. Interaction with acetylated carbohydrates differs from the one previously described for L-ficolin. This study also reveals the structural determinants for binding to sialylated compounds, a property restricted to human M-ficolin and its mouse counterpart, ficolin B. Finally, comparison between the ligand-bound structures obtained at neutral pH and nonbinding conformations observed at pH 5.6 reveals how the ligand binding site is dislocated at acidic pH. This means that the binding function of M-ficolin is subject to a pH-sensitive conformational switch. Considering that the homologous ficolin B is found in the lysosomes of activated macrophages (Runza, V. L., Hehlgans, T., Echtenacher, B., Zahringer, U., Schwaeble, W. J., and Mannel, D. N. (2006) J. Endotoxin Res. 12, 120-126), we propose that this switch could play a physiological role in such acidic compartments.     

Liver-specific knockdown of JNK1 up-regulates proliferator-activated receptor gamma coactivator 1 beta and increases plasma triglyceride despite reduced glucose and insulin levels in diet-induced obese mice

The c-Jun N-terminal kinases (JNKs) have been implicated in the development of insulin resistance, diabetes, and obesity. Genetic disruption of JNK1, but not JNK2, improves insulin sensitivity in diet-induced obese (DIO) mice. We applied RNA interference to investigate the specific role of hepatic JNK1 in contributing to insulin resistance in DIO mice. Adenovirus-mediated delivery of JNK1 short-hairpin RNA (Ad-shJNK1) resulted in almost complete knockdown of hepatic JNK1 protein without affecting JNK1 protein in other tissues. Liver-specific knockdown of JNK1 resulted in significant reductions in circulating insulin and glucose levels, by 57 and 16%, respectively. At the molecular level, JNK1 knockdown mice had sustained and significant increase of hepatic Akt phosphorylation. Furthermore, knockdown of JNK1 enhanced insulin signaling in vitro. Unexpectedly, plasma triglyceride levels were robustly elevated upon hepatic JNK1 knockdown. Concomitantly, expression of proliferator-activated receptor gamma coactivator 1 beta, glucokinase, and microsomal triacylglycerol transfer protein was increased. Further gene expression analysis demonstrated that knockdown of JNK1 up-regulates the hepatic expression of clusters of genes in glycolysis and several genes in triglyceride synthesis pathways. Our results demonstrate that liver-specific knockdown of JNK1 lowers circulating glucose and insulin levels but increases triglyceride levels in DIO mice.     

An alternative approach to normalization and evaluation for gait patterns: Procrustes analysis applied to the cyclograms of sprinters and middle-distance runners

In order to compare gait patterns, a common procedure is to normalize strides both in time and magnitude. The stride duration is usually normalized to a time percentage before averaging curves. As the timing of event occurrences may shift across strides, the shape of the averaged curves is distorted and therefore the standard deviation is overvalued. Stride magnitude normalization is performed by means of dimensionless numbers. However, there is little agreement on which body size correction methods should be used. The Procrustes method describes curve shape and shape change in a mathematical and statistical framework, independently of time and size factors. The present study aims to explore how this technique may be used for time- and magnitude-stride normalization to reflect individual and group mean responses. The Procrustes method, which combines quantitative and visual features, is applied to the shape of the ankle and knee cyclograms. Superimposition of 25 cyclograms (10 for sprinters (SP) and 15 for middle-distance runners (MDR)) was supplemented by statistical procedures (principal component analysis, discriminant function) to extract the main key events, which vary according to the athletic specialities. In comparison with the MDR (poulaine-shaped cyclogram), the ovoid cyclogram of SP reveals the following gait indicators: a short braking phase, a rapid initial lower limb swing in the forward direction, a fast upward movement of the knee and ankle, and an active foot contact. The Procrustes approach could be used to describe other quasi-periodic movements through relative motion plots (e.g., cyclograms, angle-angle diagrams, phase plane portraits).     

MRI-derived body segment parameters of children differ from age-based estimates derived using photogrammetry

Body segment parameters are required when researching joint kinetics using inverse dynamics models. However, the only regression equations for estimating pediatric body segment parameters across a wide age range were developed, using photogrammetry, based on 12 boys and have not been validated to date (Jensen, R.K., 1986. Body segment mass, radius and radius of gyration proportions of children. Journal of Biomechanics 19, 359–368). To assess whether these equations could validly be applied to girls, we asked whether body segment parameters estimated by the equations differ from parameters measured using a validated magnetic resonance imaging (MRI) method. If so, do the differences cause significant differences in joint kinetics during normal gait? Body segment parameters were estimated from axial MRIs of the left thigh and shank of 10 healthy girls (9.6±0.9 years) and compared to those from Jensen's equations. Kinematics and kinetics were collected for 10 walking trials. Extrema in hip and knee moments and powers were compared between the two sets of body segment parameters. With the exception of the shank mass center and radius of gyration, body segment parameters measured using MRI were significantly different from those estimated using regression equations. These systematic differences in body segment parameters resulted in significant differences in sagittal-plane joint moments and powers during gait. Nevertheless, it is doubtful that even the greatest differences in kinetics are practically meaningful (0.3%BW×HT and 0.7%BW×HT/s for moments and power at the hip, respectively). Therefore, body segment parameters estimated using Jensen's regression equations are a suitable substitute for more detailed anatomical imaging of 8–10-year-old girls when quantifying joint kinetics during gait.     

DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur

Cleavage of Notch by furin is required to generate a mature, cell surface heterodimeric receptor that can be proteolytically activated to release its intracellular domain, which functions in signal transduction. Current models propose that ligand binding to heterodimeric Notch (hNotch) induces a disintegrin and metalloprotease (ADAM) proteolytic release of the Notch extracellular domain (NECD), which is subsequently shed and/or endocytosed by DSL ligand cells. We provide evidence for NECD release and internalization by DSL ligand cells, which, surprisingly, did not require ADAM activity. However, losses in either hNotch formation or ligand endocytosis significantly decreased NECD transfer to DSL ligand cells, as well as signaling in Notch cells. Because endocytosis-defective ligands bind hNotch, but do not dissociate it, additional forces beyond those produced through ligand binding must function to disrupt the intramolecular interactions that keep hNotch intact and inactive. Based on our findings, we propose that mechanical forces generated during DSL ligand endocytosis function to physically dissociate hNotch, and that dissociation is a necessary step in Notch activation.     

Knockdown and mortality comparisons among spinosad-, imidacloprid-, and methomyl-containing baits against susceptible Musca domestica (Diptera: Muscidae) under laboratory conditions

The activity of spinosad, imidacloprid, and methomyl baits and technical actives were assessed against susceptible house flies, Musca domestica L. (Diptera: Muscidae). In a feeding assay, imidacloprid affected flies more rapidly than methomyl or spinosad, but spinosad was 2.7 times more potent than methomyl and 8 times more potent than imidacloprid. The profile of technical actives correlated with their respective fly bait formulations in laboratory assays. Although having the most rapid onset of activity in laboratory tests, up to 50% of flies remained alive after exposure to imidacloprid bait. In contrast, <5% of flies survived 24-h exposure to spinosad or methomyl baits. High temperature reduced the knockdown activity of imidacloprid bait and slowed the speed of kill for spinosad and methomyl baits over a 24-h exposure period. Spinosad and methomyl baits were also superior to imidacloprid when applied to the floors of environmentally controlled rooms at label recommended rates, providing good fly control for up to 21 d. The fact that a significant percentage of flies exposed to imidacloprid were rapidly knocked down but subsequently remained alive in all of the assays suggested that flies were recovering from initial exposure to this compound. Given its favorable safety profile, a high degree of initial and residual activity comparable with methomyl and lack of cross-resistance to other chemistries, spinosad bait may be a valuable component of house fly control programs to help control or delay the emergence of resistant populations.     

Bcl-2 expression modulates cell adhesion and migration promoting branching of ureteric bud cells

Bcl-2 is the founding member of a family of proteins that influence apoptosis. During kidney development bcl-2 not only acts as a survival factor, but may also impact cell adhesive mechanisms and by extension branching morphogenesis. The interrelationship between cell adhesion, migration and apoptosis, important during development, is poorly understood. Here we examined the impact lack of bcl-2, an inhibitor of apoptosis, has on ureteric bud (UB) cell adhesion, migration, and branching morphogenesis. Bcl-2 -/- UB cells demonstrated increased cell migration, increased cell invasion and decreased adhesion to vitronectin and fibronectin compared with wild-type cells. Bcl-2 +/+ UB cells readily branched in collagen gel and Matrigel while bcl-2 -/- UB cells did not undergo significant branching in either matrix. Re-expression of bcl-2 in bcl-2 -/- UB cells restored their ability to undergo branching morphogenesis in Matrigel. Consistent with our in vitro data, we show that in the absence of bcl-2, embryonic kidneys undergo decreased UB branching. We observed decreased numbers of UB branch points, UB branch tips and a decreased distance to the first UB branch point in the absence of bcl-2. The alterations in bcl-2 -/- UB cell adhesion and migration was also associated with a significant alteration in expression of a number of extracellular matrix proteins. Bcl-2 -/- UB cells exhibited increased fibronectin expression and decreased thrombospondin-1 and osteopontin expression. Taken together, these data suggest that bcl-2 is required for the proper regulation of cell adhesive and migratory mechanisms, perhaps through modulation of the cellular microenvironment.     

TetraploidMap for Windows: linkage map construction and QTL mapping in autotetraploid species

An earlier program, TetraploidMap, enabled linkage analysis to be performed for autotetraploid species, with a text-based input and output. The current program, TetraploidMap for Windows, is considerably enhanced, and now goes beyond linkage analysis to perform quantitative trait locus (QTL) interval mapping, with a range of models and thresholds assessed by permutation tests. A Windows-based interface facilitates data entry and exploration. TetraploidMap for Windows is freely available from the Web site of Bioinformatics and Statistics Scotland at http://www.bioss.ac.uk/ (user-friendly software).     

Necrotic tumor cell death in vivo impairs tumor-specific immune responses

The manner in which cells die is believed to have a major impact on the nature of immune responses to their released Ags. In this study, we present the first direct analysis of tumor-specific immune responses to in vivo occurring tumor cell death through apoptosis or necrosis. Mice bearing thymidine kinase-transfected tumors were treated either with ganciclovir to induce tumor cell apoptosis in vivo or a vascular targeting agent, ZD6126, to induce tumor cell necrosis in vivo. In contrast to tumor apoptosis, induction of necrosis reduced the frequency and impaired the function of tumor-specific CD8(+) T cells. Adoptive transfer of lymphocytes from mice with apoptotic tumors into tumor-challenged mice resulted in a significant tumor protection, which was absent when splenocytes were transferred from mice with necrotic tumors. Anti-CD40 treatment reversed impaired Ag-specific CD8(+) T cell responses in these mice. These observations have not only fundamental importance for the development of immunotherapy protocols but also help to understand the underlying mechanism of in vivo immune responses to tumor cell death.     

Inhibition of alpha interferon signaling by hepatitis B virus

Alpha interferon (IFN-alpha) and pegylated IFN-alpha (pegIFN-alpha) are used for the treatment of chronic hepatitis B (CHB). Unfortunately, only a minority of patients can be cured. The mechanisms responsible for hepatitis B virus (HBV) resistance to pegIFN-alpha treatment are not known. pegIFN-alpha is also used to treat patients with chronic hepatitis C (CHC). As with chronic hepatitis B, many patients with chronic hepatitis C cannot be cured. In CHC, IFN-alpha signaling has been found to be inhibited by an upregulation of protein phosphatase 2A (PP2A). PP2A inhibits protein arginine methyltransferase 1 (PRMT1), the enzyme that catalyzes the methylation of the important IFN-alpha signal transducer STAT1. Hypomethylated STAT1 is less active because it is bound by its inhibitor, PIAS1. In the present work, we investigated whether similar molecular mechanisms are also responsible for the IFN-alpha resistance found in many patients with chronic hepatitis B. We analyzed the expression of PP2A, the enzymatic activity of PRMT1 (methylation assays), the phosphorylation and methylation of STAT1, the association of STAT1 with PIAS1 (via coimmunoprecipitation assays), the binding of activated STAT1 to interferon-stimulated response elements (via electrophoretic mobility shift assays), and the induction of interferon target genes (via real-time RT-PCR) in human hepatoma cells expressing HBV proteins as well as in liver biopsies from patients with chronic hepatitis B and from controls. We found an increased expression of PP2A and an inhibition of IFN-alpha signaling in cells expressing HBV proteins and in liver biopsies of patients with CHB. The molecular mechanisms involved are similar to those found in chronic hepatitis C.