A PAF-acetylhydrolase activity in Tetrahymena pyriformis cells

AbstractOur study provides evidence for the existence of an acylhydrolase activity in Tetrahymena pyriformis cells, capable of hydrolizing the sn-2 ester bond of the PAF molecule. This activity is mainly distributed in the microsomal fraction (76.5% of total) and has properties similar to the mammalian PAF-acetylhydrolase since it is Ca2+-independent, acid-labile, is inhibited by DFP and PMSF but it is not affected by egg yolk phosphatidylcholine. This microsomal acylhydrolase has apparent Km and Vmax values of 1.56 μM and 373 pmols - mg - min respectively. This is the first report of the existence of a PAF-acetylhydrolase activity in a non-mammalian cell

Enrichment of Olive Oils with Natural Bioactive Compounds from Aromatic and Medicinal Herbs: Phytochemical Analysis and Antioxidant Potential

Olive oil and herbs, two key components of the Mediterranean diet, are known for their beneficial effects on humans. In our study, we incorporated aromatic and medicinal herbs into local monovarietal olive oils via maceration procedures for enrichment. We identified the herbal-derived ingredients that migrate to olive oils and contribute positively to their total phenolic content and functional properties, such as radical scavenging activity. Thus, we characterized the essential oil composition of the aromatic herbs (GC-MS), and we determined the phenolic content and antioxidant capacity of the additives and the virgin olive oils before and after enrichment. The herbal phenolic compounds were analyzed by LC-LTQ/Orbitrap HRMS. We found that olive oils infused with Origanum vulgare ssp. hirtum, Rosmarinus officinalis and Salvia triloba obtained an increased phenolic content, by approximately 1.3 to 3.4 times, in comparison with the untreated ones. Infusion with S. triloba led to a significantly higher antioxidant capacity. Rosmarinic acid, as well as phenolic glucosides, identified in the aromatic herbs, were not incorporated into olive oils due to their high polarity. In contrast, phenolic aglycones and diterpenes from R. officinalis and S. triloba migrated to the enriched olive oils, leading to a significant increase in their phenolic content and to an improvement in their free radical scavenging capacity

Docosahexaenoic Acid Inhibits Proliferation of EoL-1 Leukemia Cells and Induces Cell Cycle Arrest and Cell Differentiation

Τhe effect of docosahexaenoic acid (DHA, an omega-3 polyunsaturated fatty acid) upon the proliferation of EoL-1 (Eosinophilic leukemia) cell line was assessed, while additional cellular events during the antiproliferative action were recorded. DHA inhibited EoL-1 cells growth dose-dependently by inducing growth arrest at G0/1 phase of the cell cycle. After DHA addition to the cells, the expression of MYC oncogene was decreased, PTAFR-mRNA overexpression was observed which was used as a marker of differentiation, and PLA2G4A-mRNA increase was recorded. The enzymatic activities of phospholipase A2 (PLA2), a group of hydrolytic enzymes, whose action precedes and leads to PAF biosynthesis through the remodeling pathway, as well as platelet activating factor acetylhydrolase (PAFAH) which hydrolyses and deactivates PAF, were also measured. DHA had an effect on the levels of both the intracellular and secreted activities of PLA2 and PAFAH. The inflammatory cytokines IL-6 and TNF-α were also detected in high levels. In conclusion, DHA-induced EoL-1 cells differentiation was correlated with downregulation of MYC oncogene, overexpression of PTAFR and PLA2G4A-mRNAs, increase of the inflammatory cytokines production, and alteration of the enzymatic activities that regulate PAF levels. DHA is a natural substance and the understanding of its action on EoL-1 cells on molecular level could be useful in further investigation as a future therapeutic tool against F/P + hypereosinophilic syndrome

Effect of azithromycin on the LPS-induced production and secretion of phospholipase A2 in lung cells

AbstractAzithromycin is a member of macrolides, utilized in the treatment of infections. Independently, these antibiotics also possess anti-inflammatory and immunomodulatory properties. Phospholipase A2 isotypes, which are implicated in the pathophysiology of inflammatory lung disorders, are produced by alveolar macrophages and other lung cells during inflammatory response and can promote lung injury by destructing lung surfactant. The aim of the study was to investigate whether in lung cells azithromycin can inhibit secretory and cytosolic phospholipases A2, (sPLA2) and (cPLA2), respectively, which are induced by an inflammatory trigger. In this respect, we studied the lipopolysaccharide (LPS)-mediated production or secretion of sPLA2 and cPLA2 from A549 cells, a cancer bronchial epithelial cell line, and alveolar macrophages, isolated from bronchoalveolar lavage fluid of ARDS and control patients without cardiopulmonary disease or sepsis. Pre-treatment of cells with azithromycin caused a dose-dependent decrease in the LPS-induced sPLA2-IIA levels in A549 cells. This inhibition was rather due to reduced PLA2G2A mRNA expression and secretion of sPLA2-IIA protein levels, as observed by western blotting and indirect immunofluorescence by confocal microscopy, respectively, than to the inhibition of the enzymic activity per se. On the contrary, azithromycin had no effect on the LPS-induced production or secretion of sPLA2-IIA from alveolar macrophages. The levels of LPS-induced c-PLA2 were not significantly affected by azithromycin in either cell type. We conclude that azithromycin exerts anti-inflammatory properties on lung epithelial cells through the inhibition of both the expression and secretion of LPS-induced sPLA2-IIA, while it does not affect alveolar macrophages

Red Blood Cell Proteasome in Beta-Thalassemia Trait: Topology of Activity and Networking in Blood Bank Conditions

Proteasomes are multi-catalytic complexes with important roles in protein control. Their activity in stored red blood cells (RBCs) is affected by both storage time and the donor’s characteristics. However, apart from their abundancy in the membrane proteome, not much is known about their topology, activity, and networking during the storage of RBCs from beta-thalassemia trait donors (beta Thal(+)). For this purpose, RBC units from fourteen beta Thal(+) donors were fractionated and studied for proteasome activity distribution and interactome through fluorometric and correlation analyses against units of sex- and aged-matched controls. In all the samples examined, we observed a time-dependent translocation and/or activation of the proteasome in the membrane and a tight connection of activity with the oxidative burden of cells. Proteasomes were more active in the beta Thal(+) membranes and supernatants, while the early storage networking of 20S core particles and activities showed a higher degree of connectivity with chaperones, calpains, and peroxiredoxins, which were nonetheless present in all interactomes. Moreover, the beta Thal(+) interactomes were specially enriched in kinases, metabolic enzymes, and proteins differentially expressed in beta Thal(+) membrane, including arginase-1, piezo-1, and phospholipid scramblase. Overall, it seems that beta Thal(+) erythrocytes maintain a considerable “proteo-vigilance” during storage, which is closely connected to their distinct antioxidant dynamics and membrane protein profile

Deciphering the Relationship Between Free and Vesicular Hemoglobin in Stored Red Blood Cell Units

Red blood cells (RBCs) release hemoglobin (Hb)-containing extracellular vesicles (EVs) throughout their lifespan in the circulation, and especially during senescence, by spleen-facilitated vesiculation of their membrane. During ex vivo aging under blood bank conditions, the RBCs lose Hb, both in soluble form and inside EVs that accumulate as a part of storage lesion in the supernatant of the unit. Spontaneous hemolysis and vesiculation are increasingly promoted by the storage duration, but little is known about any physiological linkage between them. In the present study, we measured the levels of total extracellular and EV-enclosed Hb (EV-Hb) in units of whole blood (n = 36) or packed RBCs stored in either CPDA-1 (n = 99) or in CPD-SAGM additive solution (n = 46), in early, middle, and late storage. The spectrophotometry data were subjected to statistical analysis to detect possible correlation(s) between storage hemolysis and EV-Hb, as well as the threshold (if any) that determines the area of this dynamic association. It seems that the percentage of EV-Hb is negatively associated with hemolysis levels from middle storage onward by showing low to moderate correlation profiles in all strategies under investigation. Moreover, 0.17% storage hemolysis was determined as the potential cut-off, above which this inverse correlation is evident in non-leukoreduced CPDA units. Notably, RBC units with hemolysis levels > 0.17% are characterized by higher percentage of nanovesicles (<100 nm) over typical microvesicles (100-400 nm) compared with the lower hemolysis counterparts. Our results suggest an ordered loss of Hb during RBC accelerated aging that might fuel targeted research to elucidate its mechanistic basis