Inhibition of palmitate‐induced GADD34 expression augments apoptosis in mouse insulinoma cells (MIN6)

Saturated fatty acids like palmitate induce endoplasmic reticulum (ER) stress in pancreatic beta‐cells, an event linked to apoptotic loss of β‐cells in type 2 diabetes. Sustained activation of the ER stress response leads to expression of growth arrest and DNA damage‐inducible protein 34 (GADD34), a regulatory subunit of protein phosphatase 1. In the present study, we have used small interfering RNA in order to knockdown GADD34 expression in insulin‐producing MIN6 cells prior to induction of ER stress by palmitate and evaluated its consequences on RNA‐activated protein kinase‐like ER‐localized eIF2alpha kinase (PERK) signalling and apoptosis. Salubrinal, a specific inhibitor of eukaryotic initiation factor 2α (eIF2α) dephosphorylation, was used as a comparison. Salubrinal treatment augmented palmitate‐induced ER stress and increased GADD34 levels. Both GADD34 knockdown and salubrinal treatment potentiated the cytotoxic effects of palmitate as evidenced by increased DNA fragmentation and activation of caspase 3, with the fundamental difference that the former did not involve enhanced levels of GADD34. The data from this study suggest that sustained activation of PERK signalling and eIF2α phosphorylation sensitizes insulin‐producing MIN6 cells to lipoapoptosis independently of GADD34 expression levels. Copyright © 2014 John Wiley & Sons, Ltd.     

Environment versus dispersal in the assembly of western Amazonian palm communities

Aim It is a central issue in ecology and biogeography to understand what governs community assembly and the maintenance of biodiversity in tropical rain forest ecosystems. A key question is the relative importance of environmental species sorting (niche assembly) and dispersal limitation (dispersal assembly), which we investigate using a large dataset from diverse palm communities. Location Lowland rain forest, western Amazon River Basin, Peru. Methods We inventoried palm communities, registering all palm individuals and recording environmental conditions in 149 transects of 5 m × 500 m. We used ordination, Mantel tests and indicator species analysis (ISA) to assess compositional patterns, species responses to geographical location and environmental factors. Mantel tests were used to assess the relative importance of geographical distance (as a proxy for dispersal limitation) and environmental differences as possible drivers of dissimilarity in palm species composition. We repeated the Mantel tests for subsets of species that differ in traits of likely importance for habitat specialization and dispersal (height and range size). Results We found a strong relationship between compositional dissimilarity and environmental distance and a weaker but also significant relationship between compositional dissimilarity and geographical distance. Consistent with expectations, relationships with environmental and geographical distance were stronger for understorey species than for canopy species. Geographical distance had a higher correlation with compositional dissimilarity for small‐ranged species compared with large‐ranged species, whereas the opposite was true for environmental distance. The main environmental correlates were inundation and soil nutrient levels. Main conclusions The assembly of palm communities in the western Amazon appears to be driven primarily by species sorting according to hydrology and soil, but with dispersal limitation also playing an important role. The importance of environmental characteristics and geographical distance varies depending on plant height and geographical range size in agreement with functional predictions, increasing our confidence in the inferred assembly mechanisms.     

Properties of SDS-polyacrylamide gels highly cross-linked with N,N'-diallyltartardiamide and the rapid isolation of macromolecules from the gel matrix.

Polyacrylamide gels cross-linked with N,N′-diallyltartardiamide (DATD) are, in contrast to gels cross-linked with N,N′-methylenebisacrylamide (Bis), readily solubilized by periodic acid (Anker, H. S., 1970, FEBS Lett.7, 293), thus permitting efficient analyses of electrophoretically separated, labeled biological material. The capacities of polyacrylamide gels, cross-linked with Bis and DATD, to serve as media for electrophoretic separation of proteins, were compared. As DATD-cross-linked gels were inferior to equimolar Bis-crosslinked gels with 5% cross-linking (C_Bis = 5%) by the criteria of more pronounced swelling, markedly softer gels and, less concentrated and bended protein zones on electrophoresis and subsequent staining, gels cross-linked with different percentage CDATD were examined. The water regain of DATD-cross-linked gels, the retardation coefficients, and free mobilities of different proteins in equimolar Bis- and DATD-cross-linked gels were determined. When the DATD concentration in gels was increased to C_DATD = 27%, gels assumed physical characteristics comparable to those cross-linked with Bis at C_Bis = 5%. We report further the rapid, facile isolation of protein bands out of the gel matrix cross-linked with DATD. However, the isolation procedure results in an irreversible loss of biological activity.     

Iron-sulfur centers in the photosynthetic reaction center complex fromChlorobium vibrioforme. Differences from and similarities to the iron-sulfur centers in Photosystem I

The photosynthetic reaction center complex from the green sulfur bacteriumChlorobium vibrioforme has been isolated under anaerobic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals polypeptides with apparent molecular masses of 80, 40, 30, 18, 15, and 9 kDa. The 80- and 18-kDa polypeptides are identified as the reaction center polypeptide and the secondary donor cytochromec ₅₅₁ encoded by thepscA andpscC genes, respectively. N-terminal amino acid sequences identify the 40-kDa polypeptide as the bacteriochlorophylla-protein of the baseplate (the Fenna-Matthews-Olson protein) and the 30-kDa polypeptide as the putative 2[4Fe-4S] protein encoded bypscB. Electron paramagnetic resonance (EPR) analysis shows the presence of an iron-sulfur cluster which is irreversibly photoreduced at 9K. Photoaccumulation at higher temperature shows the presence of an additional photoreduced cluster. The EPR spectra of the two iron-sulfur clusters resemble those of F_A and F_B of Photosystem I, but also show significantly differentg-values, lineshapes, and temperature and power dependencies. We suggest that the two centers are designated Center I (with calculatedg-values of 2.085, 1.898, 1.841), and Center II (with calculatedg-values of 2.083, 1.941, 1.878). The data suggest that Centers I and II are bound to thepscB polypeptide.     

Optimization of 2-piperidin-4-yl-acetamides as melanin-concentrating hormone receptor 1 (MCH-R1) antagonists: Designing out hERG inhibition

Herein, we disclose the discovery and optimization of 2-piperidin-4-yl-acetamide derivatives as MCH-R1 antagonists. Structural investigation of piperidin-4-yl-amide and piperidin-4-yl-ureas identified 2-piperidin-4-yl-acetamide-based MCH-R1 antagonists with outstanding in vivo efficacy but flawed with high affinity towards the hERG potassium channel. While existing hERG SAR information was employed to discover highly potent MCH-R1 antagonists with minimized hERG inhibition, additional hurdles prevented their subsequent clinical exploration.     

Solubilization of immune precipitates by complement in the absence of properdin or factor D

Various experiments have demonstrated that immune precipitates (IPs) are not solubilized by complement in the absence of alternative pathway function. To determine whether the characteristics of the IPs were responsible for these observations, we studied the solubilization (Sol) of IPs formed by bovine serum albumin (BSA)-rabbit antiBSA and tetanus toxoid (TT)-human antiTT. Sera deficient in properdin solubilized a fraction of BSA-antiBSA precipitates, although only when the IPs were formed in antibody excess. The same sera solubilized TT-antiTT precipitates with some delay but almost as efficiently as normal serum. Factor D-depleted serum solubilized a fraction of TT-antiTT precipitates too, indicating that Sol may proceed through activation of the classical pathway only. Thus, the requirements for complement-mediated Sol depend on the characteristics of the IPs and do not necessarily include alternative pathway function.