Presence Of Phosphatidylserine Research Articles

Macrophages and Natural Killers Degrade α-Synuclein Aggregates.

Amyloid oligomers and fibrils are protein aggregates that exert a high cell toxicity. Efficient degradation of these protein aggregates can minimize the spread and progression of neurodegeneration. In this study, we investigate the properties of natural killer (NK) cells and macrophages in the degradation of α-synuclein (α-Syn) aggregates grown in a lipid-free environment and in the presence of phosphatidylserine and cholesterol (PS/Cho), which are lipids that are directly associated with the onset and progression of Parkinson's disease. We found that both types of α-Syn aggregates were endocytosed by neurons, which caused strong damage to cell endosomes. Our results also indicated that PS/Cho vesicles drastically increased the toxicity of α-Syn fibrils formed in their presence compared to the toxicity of α-Syn aggregates grown in a lipid-free environment. Both NK cells and macrophages were able to degrade α-Syn and α-Syn/Cho monomers, oligomers, and fibrils. Quantitative analysis of protein degradation showed that macrophages demonstrated substantially more efficient internalization and degradation of amyloid aggregates in comparison to NK cells. We also found that amyloid aggregates induced the proliferation of macrophages and NK cells and significantly changed the expression of their cytokines and chemokines.

Abstract 4606: Combinatorial effects of a sequence of trabectedin (T) and CD13-targeted tissue factor tTF-NGR on human vascular endothelial cells (HUVEC) and in soft tissue sarcoma (STS) models

Abstract Trabectedin represents a standard treatment option for relapsed or refractory soft tissue sarcomas. CD13 is a neutral aminopeptidase expressed on remodeling and invasive endothelial cells such as in the tumor vasculature. CD13-targeted tissue factor (tTF-NGR) is a recombinant pro-coagulatory fusion protein which accumulates in the tumor vasculature leading to tumor vascular occlusion and ultimately to tumor infarction. Giving both compounds in sequence could trap trabectedin inside tumors and thus increase its efficacy. This vice versa can optimize the activity of tTF-NGR. To prepare a multicenter clinical trial of a combination of trabectedin and tTF-NGR in patients with advanced soft tissue sarcomas refractory to 1st line systemic therapy (TRABTRAP), preclinical experiments on the mechanism of action of this combination, its efficacy and pharmacokinetics (PK) were performed. We first tested the pro-apoptotic function of trabectedin on human vascular endothelial cells (HUVEC) and human HT1080 sarcoma cells in vitro. Trabectedin significantly (p<.0001) increased the presence of phosphatidylserine (PS) on the outer leaflet of the phospholipid bilayer of the cell membrane in a dose- and time-dependent manner. The resulting optimized phospholipid milieu significantly potentiated the pro-coagulatory efficacy of tTF-NGR within the factor X:factor VIIa:tTF-NGR:CD13 complex on the outer cell membrane when compared to cultures without trabectedin (p<.05). This effect was specifically dependent on the presence of PS as it could be abolished by masking PS via preincubation with annexin V, whereas corticosteroids had no effect on the pro-coagulatory response of HUVEC in the presence of tTF-NGR. In an in vivo HT1080 human sarcoma xenograft model in athymic mice, systemic combination treatment of trabectedin followed by tTF-NGR (n=11) inhibited tumor growth to a greater extent than either compound alone (trabectedin n=9, tTF-NGR n=9) and compared to a saline control (n=8). The terminal half-life of tTF-NGR infused intravenously over 1 hour, was 9 hours (phase I trial). To further characterize the duration of tTF-NGR presence upon binding to the endothelial cell surface molecule CD13, we performed flow cytometry and fluorescence labeling experiments with tTF-NGR and HUVEC in vitro. After coincubation with HUVEC, the cells rapidly internalized the tTF-NGR:CD13 complex into the cytoplasm of the cells (approx. 50% of the labeled tTF-NGR after 3 h at 37oC), a process which exposed the protein to intracellular degradation. The multicenter TRABTRAP trial is actively recruiting patients (EudraCT 2020-005858-21). Citation Format: Caroline Brand, Stefanie Pavelka, Kathrin Hessling, Heike Hintelmann, Andrew F. Berdel, Sebastian Bäumer, Nicole Bäumer, Georg Lenz, Christoph Schliemann, Wolfgang E. Berdel, Christian Schwöppe. Combinatorial effects of a sequence of trabectedin (T) and CD13-targeted tissue factor tTF-NGR on human vascular endothelial cells (HUVEC) and in soft tissue sarcoma (STS) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4606.

Role of Saturation and Length of Fatty Acids of Phosphatidylserine in the Aggregation of Transthyretin.

The progressive accumulation of transthyretin (TTR), a small protein that transports thyroxine, in various organs and tissues is observed upon transthyretin amyloidosis, a severe pathology that affects the central, peripheral, and autonomic nervous systems. Once expressed in the liver and choroid plexus, TTR is secreted into the bloodstream and cerebrospinal fluid. In addition to thyroxine, TTR interacts with a large number of molecules, including retinol-binding protein and lipids. In this study, we examined the extent to which phosphatidylserine (PS), a phospholipid that is responsible for the recognition of apoptotic cells by macrophages, could alter the stability of TTR. Using thioflavin T assay, we investigated the rates of TTR aggregation in the presence of PS with different lengths and saturation of fatty acids (FAs). We found that all analyzed lipids decelerated the rate of TTR aggregation. We also used a set of biophysical methods to investigate the extent to which the presence of PS altered the morphology and secondary structure of TTR aggregates. Our results showed that the length and saturation of fatty acids in PS uniquely altered the morphology and secondary structure of TTR fibrils. As a result, TTR fibrils that were formed in the presence of PS with different lengths and saturation of FAs exerted significantly lower cell toxicity compared with the TTR aggregates grown in the lipid-free environment. These findings help to reveal the role of PS in transthyretin amyloidosis and determine the role of the length and saturation of FAs in PS on the morphology and secondary structure of TTR fibrils.

Length and saturation of fatty acids in phosphatidylserine determine the rate of lysozyme aggregation simultaneously altering the structure and toxicity of amyloid oligomers and fibrils.

Abrupt aggregation of misfolded proteins is the underlying molecular cause of numerous severe pathologies including Alzheimer's and Parkinson's diseases. Protein aggregation yields small oligomers that can later propagate into amyloid fibrils, β-sheet-rich structures with a variety of topologies. A growing body of evidence suggests that lipids play an important role in abrupt aggregation of misfolded proteins. In this study, we investigate the roles of length and saturation of fatty acids (FAs) in phosphatidylserine (PS), an anionic lipid that is responsible for the recognition of apoptotic cells by macrophages, in lysozyme aggregation. We found that both the length and saturation of FAs in PS contribute to the aggregation rate of insulin. PS with 14-carbon-long FAs (14:0) enabled a much stronger acceleration of protein aggregation compared to PS with 18-carbon-long FAs (18:0). Our results demonstrate that the presence of double bonds in FAs accelerated the rate of insulin aggregation relative to PS with fully saturated FAs. Biophysical methods revealed morphological and structural differences in lysozyme aggregates grown in the presence of PS with varying lengths and FA saturation. We also found that such aggregates exerted diverse cell toxicities. These results demonstrate that the length and saturation of FAs in PS can uniquely alter the stability of misfolded proteins on lipid membranes.

대장균에서 발현된 넙치 PKCβ1의 활성도 조사

Oliver flounder (Paralichthys olivaceus) is the second most farmed fish species in Korea in 2020. Thus, oliver flounder was studied on aquaculture methods, pathogenic viruses and bacteria, and gene duplication of teleostei fish have been actively conducted. Protein kinase C (PKC) is an enzyme that phosphorylates serine/threonine residues of proteins. PKC is divided to several subfamilies (conventional, novel, atypical) depending on how it is activated. PKCβ is activated by Ca2+ and diacylglycerol (DAG) in the presence of phosphatidylserine. The recombinant protein of oliver flounder PKCβ1 (PoPKCβ1) was expressed and purified, and the activity was measured in several reaction conditions such as temperature, pH, and recombinant protein concentration. As a result, it exhibited a wide range of activity from 10°C to 50°C by reaction temperature, and sufficient activity from 5 to 10 by pH. And 50 ng/μl showed the highest activity in the recombinant protein concentration. Therefore this study suggests that PoPKCβ1 can perform phosphorylation of proteins under a wide range of conditions.

Transcriptional activity of repair, apoptosis and cell cycle genes (TP53, MDM2, ATM, BAX, BCL-2, CDKN1A, OGG1, XPC, PADI4, MAPK8, NF-KB1, STAT3, GATA3) in chronically exposed persons with different intensity of apoptosis of peripheral blood lymphocytes.

Transcriptional activity of genes involved in maintaining genetic homeostasis (genes for repair, cell cycle and apoptosis: TP53, MDM2, ATM, BAX, BCL-2, CDKN1A, OGG1, XPC, PADI4, MAPK8, NF-KB1, STAT3, GATA3) was studied in chronically exposed persons with an increased intensity of early and late stages of apoptosis and necrosis of peripheral blood lymphocytes. The object of this study was peripheral blood mononuclear cells obtained from 132 chronically exposed residents of the Techa riverside villages. The mean accumulated dose to red bone marrow was 426.4 ± 48.2 mGy (1.3–2930.0 mGy), to thymus and peripheral immune organs, 58.9 ± 7.9 mGy (0.1–489.0 mGy). The study was performed more than 60 years after the onset of exposure, the average age of exposed persons was 68 ± 0.6 years (55–86 years). The study of apoptotic and necrotic death of peripheral blood lymphocytes was based on the presence of phosphatidylserine on the cell membrane surface, as well as on its permeability for DNA-intercalating dye. Evaluation of the relative content of mRNA genes for repair, cell cycle, and apoptosis was carried out using real-time PCR. An increased relative content of PADI4 gene mRNA was registered in the group of chronically exposed persons with the increased intensity of early apoptosis (p = 0.006). Modulation of the relative content of mRNA of the TP53 (p = 0.013) and BCL-2 (p = 0.021) genes was detected in the group of chronically exposed individuals with the increased intensity of the late stage of apoptosis. A statistically signif icant increase in the transcriptional activity of the TP53 gene was observed in the group of chronically exposed persons with the increased intensity of peripheral blood lymphocyte necrosis in the long-term period (p = 0.015). In the course of the study it was noted that exposed people with increased intensity of apoptosis, f irst of all, demonstrate changes in the transcriptional activity of apoptotic genes. These data are consistent with current views on the activation of programmed cell death.

DNA binding ability and cytotoxicity, cell cycle arrest and apoptosis inducing properties of a benzochromene derivative against K562 human leukemia cells

Chromene and its derivatives are generally spread in nature. Heterocylic-based compounds like chromenes have displayed pharmacological activities. Chromene derivatives are critical due to some biological features such as anticancer activity. CML, chronic myelogenous leukemia, is a fatal malignancy determined by resistance to apoptosis and contains the Philadelphia chromosome. Induction of apoptosis is one of the main approaches in cancer therapy. In this research, benzochromene derivative, 2-amino-4-(4-methoxy phenyl)-4H-benzochromene-3-carbonitrile (4-MC) was tested for cytotoxic and apoptotic induction activities in the human leukemic K562 cell line. The MTT growth inhibition assay was used to determine the cellular growth and survival. Moreover, the binding attribute of 4-MC with double helix DNA was assessed by some spectroscopic and viscosity measurement, and also for docking analysis. 4-MC exhibited good cytotoxicity on K562 cell line and the IC50 value was calculated to be 30 µM. Furthermore, the mechanisms of apoptosis induction were determined morphologically by fluorescence dual staining with acridine orange and ethidium bromide and cell cycle analysis was based on DNA content, as well as the presence of phosphatidyl serine on the outside of the cells by the flow cytometric method. The results showed that 4-MC had potent cytotoxic activity via sub-G1 cell cycle arrest and induction of apoptosis. The experimental and simulation studies reported that 4-MC binds to ctDNA through groove binding mode with the binding constant (K b) of 2.5 × 103 M−1. These data represent a considerable anticancer potential of 4-MC and could be suggested for further pharmacological studies.

Unravelling the Structural Organization of Individual α-Synuclein Oligomers Grown in the Presence of Phospholipids

Parkinson's disease (PD) is a severe neurological disorder that affects more than 1 million people in the U.S. alone. A hallmark of PD is the formation of intracellular α-synuclein (α-Syn) protein aggregates called Lewy bodies (LBs). Although this protein does not have a particular localization in the central neural system, α-Syn aggregates are primarily found in certain areas of the midbrain, hypothalamus, and thalamus. Microscopic analysis of LBs reveals fragments of lipid-rich membranes, organelles, and vesicles. These and other pieces of experimental evidence suggest that α-Syn aggregation can be triggered by lipids. In this study, we used atomic force microscope infrared spectroscopy (AFM-IR) to investigate the structural organization of individual α-Syn oligomers grown in the presence of two different phospholipids vesicles. AFM-IR is a modern optical nanoscopy technique that has single-molecule sensitivity and subdiffraction spatial resolution. Our results show that α-Syn oligomers grown in the presence of phosphatidylcholine have a distinctly different structure than oligomers grown in the presence of phosphatidylserine. We infer that this occurs because of specific charges adopted by lipids, which in turn governs protein aggregation. We also found that the protein to phospholipid ratio has a substantial impact on the structure of α-Syn oligomers. These findings demonstrate that α-Syn is far more complex than expected from the perspective of the structural organization of oligomeric species.

Апоптоз лимфоцитов и полиморфизм генов регуляции апоптоза у лиц, подвергшихся хроническому радиационному воздействию

Purpose: Study the apoptotic death of peripheral blood lymphocytes in long-term period in persons exposed to chronic radiation exposure, and analysis of association of the polymorphic regions rs4645878, rs2279115, rs28362491, rs664677, rs1042522, rs1801270, rs2279744 of the BAX, BCL2, NFkB, ATM, TP53, CDKN1A, MDM2 genes with apoptotic lymphocytes frequency in residents of the coastal villages of the Techa River.
 Material and methods: The study of apoptosis and genotyping was conducted in 390 persons exposed to chronic radiation exposure as a result of Mayak PA radioactive waste releases into the Techa–Iset–Tobol river system. The early stage of apoptosis was assessed on a flow cytometer by the presence of phosphatidylserine on the surface of the cell membranes using the Annexin V Apoptosis Detection Kit I and the late stage of apoptosis using the TUNEL method. Real-time PCR genotyping was performed of allelic variations of rs4645878, rs2279115, rs28362491, rs664677, rs1042522, rs1801270, rs2279744 of BAX, BCL2, NFkB, ATM, TP53, CDKN1A, MDM2 genes in a group of irradiated individuals.
 Results: The number of cells at the early stage of apoptosis is statistically significantly increased in individuals whose irradiation began during the period of intrauterine development and continued in the postnatal period compared to individuals exposed only in the postnatal period. At the same time, the number of lymphocytes at the stage of DNA fragmentation in the group irradiated in utero is lower than in the group irradiated postnatally and non-irradiated individuals. Also, a weak negative correlation between intrauterine doses of RBM irradiation and the doses of thymus and peripheral lymphoid organs with the number of cells in the late stage of apoptosis in individuals irradiated in utero. The influence of allelic variation rs4645878 of the BAX gene was established on the number of lymphocytes at the early stage of apoptosis in residents of coastal villages of the Techa River. A statistically significant decrease in the number of cells at an early stage of apoptosis is observed in C/C genotype carriers according to the allelic variation rs4645878 of the BAX gene compared with carriers of the T/T and T/C genotypes.
 Conclusion: Residents of coastal villages of the Techa River exposed to radiation during the period of prenatal development, there are differences in the frequency of apoptotic death of peripheral blood lymphocytes compared with non-irradiated persons and persons who were irradiated in the postnatal period. SNPs of apoptosis-regulating genes can modify the response of blood lymphocytes to radiation in a wide range of doses RBM.

Phosphatidylethanolamine-phosphatidylserine binding synergy of seven coagulation factors revealed using Nanodisc arrays on silicon photonic sensors

Blood coagulation is regulated through protein–protein and protein–lipid interactions that occur at the sub-endothelium following vascular damage. Soluble clotting proteins bind to membrane components in a phosphatidylserine (PS) dependent manner to assemble multi-protein complexes that regulate clot formation; however, PS is of limited abundance physiologically. In this manuscript, we investigate synergy between PS and phosphatidylethanolamine (PE)—a lipid of much higher abundance naturally. Using a label-free, silicon photonic technology, we constructed arrays of Nanodiscs having variable lipid composition and probed the binding interactions of seven different clotting factors with GLA domains that have never been studied in tandem experiments before. The factors studied were prothrombin, activated factor VII, factor IX, factor X, activated protein C, protein S, and protein Z. Equilibrium dissociation constants (Kd) for each coagulation factor binding to Nanodiscs with unique compositions of PE and PS were determined. While all factors showed greater binding affinities in the presence of PS and PE, the most dramatic improvements in binding were observed when PS quantities were lowest. This demonstrates that synergy is effective in promoting coagulation factor binding under physiological lipid compositions, as opposed to the artificially high PS content probed in most in vitro activity studies.

Cooperativity of membrane-protein and protein\u2013protein interactions control membrane remodeling by epsin 1 and affects clathrin-mediated endocytosis

Membrane remodeling is a critical process for many membrane trafficking events, including clathrin-mediated endocytosis. Several molecular mechanisms for protein-induced membrane curvature have been described in some detail. Contrary, the effect that the physico-chemical properties of the membrane have on these processes is far less well understood. Here, we show that the membrane binding and curvature-inducing ENTH domain of epsin1 is regulated by phosphatidylserine (PS). ENTH binds to membranes in a PI(4,5)P2-dependent manner but only induces curvature in the presence of PS. On PS-containing membranes, the ENTH domain forms rigid homo-oligomers and assembles into clusters. Membrane binding and membrane remodeling can be separated by structure-to-function mutants. Such oligomerization mutants bind to membranes but do not show membrane remodeling activity. In vivo, they are not able to rescue defects in epidermal growth factor receptor (EGFR) endocytosis in epsin knock-down cells. Together, these data show that the membrane lipid composition is important for the regulation of protein-dependent membrane deformation during clathrin-mediated endocytosis.

Aster Proteins Regulate the Accessible Cholesterol Pool in the Plasma Membrane.

Recent studies have demonstrated the existence of a discrete pool of cholesterol in the plasma membranes (PM) of mammalian cells-referred to as the accessible cholesterol pool-that can be detected by the binding of modified versions of bacterial cytolysins (e.g., anthrolysin O). When the amount of accessible cholesterol in the PM exceeds a threshold level, the excess cholesterol moves to the endoplasmic reticulum (ER), where it regulates the SREBP2 pathway and undergoes esterification. We reported previously that the Aster/Gramd1 family of sterol transporters mediates nonvesicular movement of cholesterol from the PM to the ER in multiple mammalian cell types. Here, we investigated the PM pool of accessible cholesterol in cholesterol-loaded fibroblasts with a knockdown of Aster-A and in mouse macrophages from Aster-B and Aster-A/B-deficient mice. Nanoscale secondary ion mass spectrometry (NanoSIMS) analyses revealed expansion of the accessible cholesterol pool in cells lacking Aster expression. The increased accessible cholesterol pool in the PM was accompanied by reduced cholesterol movement to the ER, evidenced by increased expression of SREBP2-regulated genes. Cosedimentation experiments with liposomes revealed that the Aster-B GRAM domain binds to membranes in a cholesterol concentration-dependent manner and that the binding is facilitated by the presence of phosphatidylserine. These studies revealed that the Aster-mediated nonvesicular cholesterol transport pathway controls levels of accessible cholesterol in the PM, as well as the activity of the SREBP pathway.

An improved and highly selective fluorescence assay for measuring phosphatidylserine decarboxylase activity

Phosphatidylserine decarboxylases (PSDs) catalyze the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a critical step in membrane biogenesis and a potential target for development of antimicrobial and anti-cancer drugs. PSD activity has typically been quantified using radioactive substrates and products. Recently, we described a fluorescence-based assay that measures the PSD reaction using distyrylbenzene-bis-aldehyde (DSB-3), whose reaction with PE produces a fluorescence signal. However, DSB-3 is not widely available and also reacts with PSD's substrate, PS, producing an adduct with lower fluorescence yield than that of PE. Here, we report a new fluorescence-based assay that is specific for PSD and in which the presence of PS causes only negligible background. This new assay uses 1,2-diacetyl benzene/β-mercaptoethanol, which forms a fluorescent iso-indole-mercaptide conjugate with PE. PE detection with this method is very sensitive and comparable with detection by radiochemical methods. Model reactions examining adduct formation with ethanolamine produced stable products of exact masses (m/z) of 342.119 and 264.105. The assay is robust, with a signal/background ratio of 24, and can readily detect formation of 100 pmol of PE produced from Escherichia coli membranes, Candida albicans mitochondria, or HeLa cell mitochondria. PSD activity can easily be quantified by sequential reagent additions in 96- or 384-well plates, making it readily adaptable to high-throughput screening for PSD inhibitors. This new assay now enables straightforward large-scale screening for PSD inhibitors against pathogenic fungi, antibiotic-resistant bacteria, and neoplastic mammalian cells.

The N-Terminal Region of a Ph-Responsive Peptide Controls Its Interaction with Phosphatidylserine-Containing Bilayers

Local acidosis is common in solid tumors, and provides a promising target for peptide-based anticancer treatments. To exploit this characteristic, the acidity-triggered rational membrane (ATRAM) peptide was designed to insert selectively into lipid bilayers when the surrounding media is acidic. Insertion of ATRAM into the bilayer as a transmembrane α-helix involves translocation of the C-terminus across the membrane, such that the N-terminus is free to interact with lipids on the outer leaflet. Since many cancers overexpose on the outer membrane leaflet the negatively charged lipid phosphatidylserine (PS), we propose that mutations at the non-inserting N-terminus would modify ATRAM's interactions with model cancer cell membranes. Here, we used two ATRAM variants, termed K2-ATRAM (L2K/L5K) and Y-ATRAM (L2Y), to explore the effects of positively charged or mildly hydrophobic amino acids near the N-terminus. The presence of PS in the membrane enhanced membrane partitioning for K2-ATRAM but decreased it for Y-ATRAM. However, screening with high-salt conditions showed that the effects of PS on the pK of insertion are multifactorial and cannot explained by electrostatics alone. These results suggest that studies using only neutral bilayers may fail to indicate the true interactions of anticancer peptides with tumor cells, which contain negatively charged phospholipids. Additionally, the different behaviors of the ATRAM variants demonstrate how the non-inserting N-terminal region can be tailored to adjust the specificity of ATRAM for target membranes. This information will aid the design of new ATRAMs and other peptides that are tailored to the pH and lipid compositions of a variety of diseases.

Sperm Lipid Composition in Early Diverged Fish Species: Internal vs. External Mode of Fertilization.

The lipid composition of sperm membranes is crucial for fertilization and differs among species. As the evolution of internal fertilization modes in fishes is not understood, a comparative study of the sperm lipid composition in freshwater representatives of externally and internally fertilizing fishes is needed for a better understanding of taxa-specific relationships between the lipid composition of the sperm membrane and the sperm physiology. The lipidomes of spermatozoa from stingray, a representative of cartilaginous fishes possessing internal fertilization, and sterlet, a representative of chondrostean fishes with external fertilization, have been studied by means of nuclear magnetic resonance (NMR), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), electrospray MS, gas chromatography-(GC) MS, and thin-layer chromatography (TLC). NMR experiments revealed higher cholesterol content and the presence of phosphatidylserine in stingray compared to sterlet sperm. Unknown MS signals could be assigned to different glycosphingolipids in sterlet (neutral glycosphingolipid Gal-Cer(d18:1/16:0)) and stingray (acidic glycosphingolipid sulpho-Gal-Cer(d18:1/16:0)). Free fatty acids in sterlet sperm indicate internal energy storage. GC-MS experiments indicated a significant amount of adrenic acid, but only a low amount of docosahexaenoic acid in stingray sperm. In a nutshell, this study provides novel data on sperm lipid composition for freshwater stingray and sterlet possessing different modes of fertilization.

Down-regulation of Bcl2 and Survivin, and up-regulation of Bax involved in copper (II) phenylthiosemicarbazone complex-induced apoptosis in leukemia stem-like KG1a cells

Previous studies suggested that phenylthiosemicarbazones are considered as a new apoptosis-inducing agent. In this study, anti-proliferative and apoptotic effects of the copper (II) phenylthiosemicarbazone complex (Cu-PTSC) were investigated in human acute myeloid leukemia KG1a cell line. The KG1a cells were treated with various concentrations (20−140 μM) of the Cu-PTSC, and cell viability was determined by MTT assay. The IC50 value of 80 ± 2.5 μM was selected for further evaluations. Apoptosis, as the antitumor strategy in the cells, was investigated morphologically by acridine orange/ethidium bromide (AO/EtBr) double staining, and surface expression assay of phosphatidylserine by Annexin V/PI technique was studied via flow cytometry. Results indicated that the cells undergo morphologic changes with chromatin condensation and G0/G1 cell cycle arrest after treatment with Cu-PTSC. The presence of phosphatidylserine on the outer surface of the cell membrane confirmed the apoptosis occurrence in the KG1a cells. Real-time PCR and western blot analyses revealed that the incubation of KG1a cells with Cu-PTSC down-regulated the expression of Bcl-2 (anti-apoptotic protein) and Survivin (as an IAP protein) while induced the expression of Bax (pro-apoptotic protein). Based on present data, it seems that Cu-PTSC may provide a novel therapeutic approach for the treatment of acute myeloid leukemia.

Monocytes mediate homing of circulating microvesicles to the pulmonary vasculature during low-grade systemic inflammation

ABSTRACT Microvesicles (MVs), a plasma membrane-derived subclass of extracellular vesicles, are produced and released into the circulation during systemic inflammation, yet little is known of cell/tissue-specific uptake of MVs under these conditions. We hypothesized that monocytes contribute to uptake of circulating MVs and that their increased margination to the pulmonary circulation and functional priming during systemic inflammation produces substantive changes to the systemic MV homing profile. Cellular uptake of i.v.-injected, fluorescently labelled MVs (J774.1 macrophage-derived) in vivo was quantified by flow cytometry in vascular cell populations of the lungs, liver and spleen of C57BL6 mice. Under normal conditions, both Ly6Chigh and Ly6Clow monocytes contributed to MV uptake but liver Kupffer cells were the dominant target cell population. Following induction of sub-clinical endotoxemia with low-dose i.v. LPS, MV uptake by lung-marginated Ly6Chigh monocytes increased markedly, both at the individual cell level (~2.5-fold) and through substantive expansion of their numbers (~8-fold), whereas uptake by splenic macrophages was unchanged and uptake by Kupffer cells actually decreased (~50%). Further analysis of MV uptake within the pulmonary vasculature using a combined model approach of in vivo macrophage depletion, ex vivo isolated perfused lungs and in vitro lung perfusate cell-based assays, indicated that Ly6Chigh monocytes possess a high MV uptake capacity (equivalent to Kupffer cells), that is enhanced directly by endotoxemia and ablated in the presence of phosphatidylserine (PS)-enriched liposomes and β3 integrin receptor blocking peptide. Accordingly, i.v.-injected PS-enriched liposomes underwent a redistribution of cellular uptake during endotoxemia similar to MVs, with enhanced uptake by Ly6Chigh monocytes and reduced uptake by Kupffer cells. These findings indicate that monocytes, particularly lung-marginated Ly6Chigh subset monocytes, become a dominant target cell population for MVs during systemic inflammation, with significant implications for the function and targeting of endogenous and therapeutically administered MVs, lending novel insights into the pathophysiology of pulmonary vascular inflammation.

Consequences of the Endogenous N-Glycosylation of Human Ribonuclease 1.

Ribonuclease 1 (RNase 1) is the most prevalent human homologue of the archetypal enzyme RNase A. RNase 1 contains sequons for N-linked glycosylation at Asn34, Asn76, and Asn88 and is N-glycosylated at all three sites in vivo. The effect of N-glycosylation on the structure and function of RNase 1 is unknown. By using an engineered strain of the yeast Pichia pastoris, we installed a heptasaccharide (Man5GlcNAc2) on the side chain of Asn34, Asn76, and Asn88 to produce the authentic triglycosylated form of human RNase 1. As a glutamine residue is not a substrate for cellular oligosaccharyltransferase, we used strategic asparagine-to-glutamine substitutions to produce the three diglycosylated and three monoglycosylated forms of RNase 1. We found that the N-glycosylation of RNase 1 at any position attenuates its catalytic activity but enhances both its thermostability and its resistance to proteolysis. N-Glycosylation at Asn34 generates the most active and stable glycoforms, in accord with its sequon being highly conserved among vertebrate species. These data provide new insight on the biological role of the N-glycosylation of a human secretory enzyme.

Eryptosis in Haemochromatosis: Implications for rheology.

Haemochromatosis is an iron-storage disease with different genetic mutations, characterized by an increased intestinal absorption of iron, resulting in a deposition of excessive amounts of iron in parenchymal cells. When the iron is released in the blood, it is left in an unliganded form, where it can participate in Haber-Weiss and Fenton reactions, creating hydroxyl radicals. Erythrocytes (RBCs) are particularly vulnerable to hydroxyl radical damage, which can result in eryptosis (programmed cell death similar to apoptosis). Here, we used flow cytometry to study the presence of eryptosis in the main genotypic variations of HFE (heterozygous and homozygous C282Y; H63D; C282Y/H63D). We also viewed RBCs from the different mutations using super-resolution Airyscan confocal microscopy. Flow cytometry showed significant changes in membrane biochemistry, indicated by the presence of phosphatidylserine (PS) proteins on the outer leaflet of the membrane, as well as increased intracellular calpain. This was found in all of the studied mutations. Airyscan fluorescence revealed PS flip and also microparticles from RBCs. Such microparticles are known to be pro-inflammatory. We conclude that RBC pathology is present in all the studied HFE mutations, even in low penetrance mutations, and this might affect rheology in these individuals.

Repurposing Fendiline as a novel anti‐viral therapeutic

The most prolonged and fatal outbreak of the Ebola virus (EBOV) occurred in 2014, claiming the lives of thousands. Although more than 40 years have passed since the initial discovery of EBOV, there are still no available treatments with FDA approval. Clinical case studies from human survivors highlighted that a hallmark of survival is a measurable immune response against specific viral proteins, such as VP40. VP40, is the canonical mediator of EBOV budding. Therefore, slowing down viral propagation through inhibiting replication, trafficking or budding could be the key to developing an efficacious treatment.VP40 interacts with host proteins and lipids to oligomerize at the plasma membrane (PM) to form the filamentous structure of the virus. The initial association between VP40 and the PM is through electrostatic and stereospecific interactions with phosphatidyl serine (PS). Following PM docking, VP40 filamentous oligomerization is also dependent upon the presence of PS at the PM. These filaments are the building block for nascent viral particles, which will ultimately bud from the cell to propagate the virus within the host. Therefore, PS within the inner leaflet of the PM is essential for viral budding and propagation. Moreover, VP40's ability to produce virus like particles (VLPs) when expressed in mammalian cells, even in the absence of the other viral proteins, allows EBOV budding to be studied in a BSL‐2 setting.Fendiline, an FDA approved drug for coronary heart disease, has been shown to significantly reduce PS from the PM. Therefore, this project focuses on repurposing Fendiline as a novel anti‐viral therapy to inhibit EBOV budding. We hypothesized that treating HEK 293 with Fendiline, would reduce VP40's ability to bind to the PM, oligomerize, and ultimately inhibit VLP production.To monitor Fendiline's effect on PS in HEK293 cells, live cell confocal imaging experiments were performed on HEK 293 cells expressing the high affinity PS probe, GFP‐LactC2. After 48 hours of treatment, 5 uM Fendiline significantly reduced GFP‐LactC2 localization at the PM by ~25% (p=0.03). Lipidomic analysis of HEK 293 cells also revealed a significant reduction in total cellular levels of PS after 48 hours of 5 uM treatment (~30%, p=0.03). To assess the effect of Fendiline on VP40 binding to the PM, HEK 293 cells expressing GFP‐VP40 were treated with Fendiline and monitored for 48 hours. Again, after 48 hours of 5 uM treatment, there was a significant reduction of GFP‐VP40 localization to the PM (~15%, p=0.0001). Functional budding assays were performed to assess the effect of Fendiline on VLP production. After 48 hours, 5 uM Fendiline reduced VLP production by ~60% (p=0.025). Scanning electron microscopy experiments revealed a significant reduction in filamentous particle length and density after 48 hours of 1 uM or 5 uM Fendiline treatment compared to untreated cells expressing FLAG‐VP40(p<0.01).Our project has highlighted that repurposing drugs such as Fendiline and targeting lipid metabolism are promising avenues for developing anti‐viral therapies. The next stop for this project is to extend the studies into live virus models in a BSL‐4 setting.Support or Funding InformationThis work has been supported by NIH training grant T32GM075762 and NIH AI081077This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.