Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by Streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by Streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis

Covalent Attachment of Proteins to Solid Supports and Surfaces via Sortase-Mediated Ligation

BACKGROUND: There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein. METHODOLOGY: Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours. CONCLUSIONS: The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays

Ideology and moral values in rhetorical framing:How wine was saved from the 19th Century Phylloxera Epidemic

Extant organizational research into crises has focused on the efforts of different actors to defend and legitimate their ideologies towards particular actions. Although insightful, such research has offered little knowledge about the moral reasoning underlying such action. In this paper, we explore how moral reasoning from different ideological viewpoints can lead to polarized debates and stalemate within the context of ecological crises. We apply our conceptual framework in an analysis of the 19th century French phylloxera epidemic. Drawing upon this analysis, we argue that, by adapting their moral reasoning, opposing stakeholder groups could maintain their underlying ideology, while at the same time pragmatically changing their actions towards the crisis. We discuss the theoretical implications of our analysis for historical research in organizational studies and research on organizations and the natural environment

Anti-Inflammatory Effect of Fluvastatin on IL-8 Production Induced by Pseudomonas aeruginosa and Aspergillus fumigatus Antigens in Cystic Fibrosis

International audienceBACKGROUND: Early in life, patients with cystic fibrosis (CF) are infected with microorganisms including bacteria and fungi, particularly Pseudomonas aeruginosa and Aspergillus fumigatus. Since recent research has identified the anti-inflammatory properties of statins (besides their lipid-lowering effects), we investigated the effect of fluvastatin on the production of the potent neutrophil chemoattractant chemokine, IL-8, in whole blood from CF patients, stimulated by Pseudomonas aeruginosa (LPS) and Aspergillus fumigatus (AFA) antigens. RESULTS: Whole blood from adult patients with CF and from healthy volunteers was collected at the Rennes University Hospital (France). Blood was pretreated for 1 h with fluvastatin (0-300 µM) and incubated for 24 h with LPS (10 µg/mL) and/or AFA (diluted 1/200). IL-8 protein levels, quantified by ELISA, were increased in a concentration-dependent manner when cells were stimulated by LPS or AFA. Fluvastatin strongly decreased the levels of IL-8, in a concentration-dependent manner, in whole blood from CF patients. However, its inhibitory effect was decreased or absent in whole blood from healthy subjects. Furthermore, the inhibition induced by fluvastatin in CF whole blood was reversed in the presence of intermediates within the cholesterol biosynthesis pathway, mevalonate, farnesyl pyprophosphate or geranylgeranyl pyrophosphate that activate small GTPases by isoprenylation. CONCLUSIONS: For the first time, the inhibitory effects of fluvastatin on CF systemic inflammation may reveal the important therapeutic potential of statins in pathological conditions associated with the over-production of pro-inflammatory cytokines and chemokines as observed during the manifestation of CF. The anti-inflammatory effect could be related to the modulation of the prenylation of signalling proteins

Correlational structure of ‘frontal’ tests and intelligence tests indicates two components with asymmetrical neurostructural correlates in old age

AbstractBoth general fluid intelligence (gf) and performance on some ‘frontal tests’ of cognition decline with age. Both types of ability are at least partially dependent on the integrity of the frontal lobes, which also deteriorate with age. Overlap between these two methods of assessing complex cognition in older age remains unclear. Such overlap could be investigated using inter-test correlations alone, as in previous studies, but this would be enhanced by ascertaining whether frontal test performance and gf share neurobiological variance. To this end, we examined relationships between gf and 6 frontal tests (Tower, Self-Ordered Pointing, Simon, Moral Dilemmas, Reversal Learning and Faux Pas tests) in 90 healthy males, aged ~73years. We interpreted their correlational structure using principal component analysis, and in relation to MRI-derived regional frontal lobe volumes (relative to maximal healthy brain size). gf correlated significantly and positively (.24≤r≤.53) with the majority of frontal test scores. Some frontal test scores also exhibited shared variance after controlling for gf. Principal component analysis of test scores identified units of gf-common and gf-independent variance. The former was associated with variance in the left dorsolateral (DL) and anterior cingulate (AC) regions, and the latter with variance in the right DL and AC regions. Thus, we identify two biologically-meaningful components of variance in complex cognitive performance in older age and suggest that age-related changes to DL and AC have the greatest cognitive impact