Adipose triglyceride lipase regulates eicosanoid production in activated human mast cells

Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A₂-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation.     

Interleukin-1α Activity in Necrotic Endothelial Cells Is Controlled by Caspase-1 Cleavage of Interleukin-1 Receptor-2: IMPLICATIONS FOR ALLOGRAFT REJECTION*

Inflammation is a key instigator of the immune responses that drive atherosclerosis and allograft rejection. IL-1α, a powerful cytokine that activates both innate and adaptive immunity, induces vessel inflammation after release from necrotic vascular smooth muscle cells (VSMCs). Similarly, IL-1α released from endothelial cells (ECs) damaged during transplant drives allograft rejection. However, IL-1α requires cleavage for full cytokine activity, and what controls cleavage in necrotic ECs is currently unknown. We find that ECs have very low levels of IL-1α activity upon necrosis. However, TNFα or IL-1 induces significant levels of active IL-1α in EC necrotic lysates without alteration in protein levels. Increased activity requires cleavage of IL-1α by calpain to the more active mature form. Immunofluorescence and proximity ligation assays show that IL-1α associates with interleukin-1 receptor-2, and this association is decreased by TNFα or IL-1 and requires caspase activity. Thus, TNFα or IL-1 treatment of ECs leads to caspase proteolytic activity that cleaves interleukin-1 receptor-2, allowing IL-1α dissociation and subsequent processing by calpain. Importantly, ECs could be primed by IL-1α from adjacent damaged VSMCs, and necrotic ECs could activate neighboring normal ECs and VSMCs, causing them to release inflammatory cytokines and up-regulate adhesion molecules, thus amplifying inflammation. These data unravel the molecular mechanisms and interplay between damaged ECs and VSMCs that lead to activation of IL-1α and, thus, initiation of adaptive responses that cause graft rejection.     

4-phenylylboronic acid: A new type of enhancer for the horseradish peroxidase catalysed chemiluminescent oxidation of luminol

4-Phenylylboronic acid enhances the light emission from the horseradish peroxidase catalysed oxidation of luminol by hydrogen peroxide. Optimization studies showed that the greatest enhancement was obtained using micromolar concentrations of the new enhancer. The largest degree of enhancement was found with the basic isoenzyme of horseradish peroxidase (Type VIA), and lesser degrees of enhancement were obtained with Type VII and Type IX horseradish peroxidase. The enhancer was also effective in the peroxidase catalysed oxidation of isoluminol by peroxide.     

Analysis of the sequence of a new cryptic plasmid, pRJF2, from a rumen bacterium of the genus Butyrivibrio: Comparison with other Butyrivibrio plasmids and application in the development of a cloning vector

Abstract A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79 bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM β1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens .     

Learning-Induced Expression of Meningeal Ependymin mRNA and Demonstration of Ependymin in Neurons and Glial Cells

Abstract: The turnover of a CNS-specific cell adhesion glycoprotein, ependymin, has earlier been found to increase during periods of neuronal plasticity. Here, ependymin mRNA expression was analyzed by semiquantitative in situ hybridization in goldfish. Learning of an active avoidance response resulted in a significant increase in ependymin mRNA expression 20 min to 4 h after acquisition of the task. In contrast, yoked control animals that were exposed to the same numbers of conditioned and unconditioned stimuli in a random, unpaired manner exhibited a strong down-regulation of ependymin mRNA. Hybridization signals were also increased by injection of anti-ependymin antiserum into brain ventricles. Ependymin mRNA was exclusively localized to reticular-shaped fibroblasts of the inner endomeningeal cell layer. Immunoelectron microscopic investigation, however, revealed ependymin also in distinct neuronal and glial cell populations in which no ependymin mRNA had been detected. Uptake of meningeal protein factors into glial and neuronal cells may therefore be of functional importance for plastic adaptations of the CNS.     

Phytoplankton and physical-chemical features of Tavropos Reservoir,Greece

Phytoplankton biomass values in Tavropos Reservoir, ranging from 92 to 1071 mg m^–3, are within a range characteristic of oligotrophic waters. The seasonal sequence of biomass shows three annual peaks, differing from the monoacmic pattern seen in oligotrophic lakes. This sequence was profoundly affected by changes in water withdrawal and inflow rates. Diatoms, cryptophytes, chrysophytes and dinoflagellates, in that order, were the major constituents of the reservoir phytoplankton. The succession, from diatoms and chrysophytes in late winter-spring, to centric diatoms in late spring-summer and again to diatom-chrysophytes in late autumn was similar to that in oligotrophic lakes.     

Mechanics of the cellular actin cortex: From signalling to shape change

The actin cortex is a thin layer of actin, myosin and actin-binding proteins that underlies the membrane of most animal cells. It is highly dynamic and can undergo remodelling on timescales of tens of seconds, thanks to protein turnover and myosin-mediated contractions. The cortex enables cells to resist external mechanical stresses, controls cell shape and allows cells to exert forces on their neighbours. Thus, its mechanical properties are the key to its physiological function. Here, we give an overview of how cortex composition, structure and dynamics control cortex mechanics and cell shape. We use mitosis as an example to illustrate how global and local regulation of cortex mechanics gives rise to a complex series of cell shape changes.