Regulation of 5-lipoxygenase by caspase-6

Das Enzym 5-Lipoxygenase (5-LO) spielt eine essentielle Rolle in der Biosynthese der Leukotriene, bioaktiver Metabolite der Arachidonsäure (AA), die an einer Vielzahl entzündlicher und allergischer Erkrankungen beteiligt sind. Die 5-LO wird bevorzugt in Zellen myeloiden Ursprungs wie Granulozyten, Monozyten oder B-Lymphozyten exprimiert. In die Regulation der zellulären 5-LO-Aktivität in der Epstein-Barr Virus-transformierten B-lymphozytären Zelllinie BL41-E95-A sind Caspasen, Aspartat-spezifische Cysteinproteasen, involviert. Das Passagieren von BL41-E95-A führt zu einer Erhöhung der Proliferationsrate der B-Lymphozyten sowie zu einem deutlichen Verlust der 5-LO-Aktivität, der mit dem Auftreten eines 62 kDa-Spaltproduktes der 5-LO und einer signifikanten Aktivitätserhöhung der Caspase-8 und -6 korreliert. Isolierte humane 5-LO wird durch rekombinante Caspase-6 zwischen Asp170 und Ser171 zu einem 58 kDa-Fragment in vitro gespalten, wobei das Tetrapeptid VEID170 innerhalb der 5-LO als Erkennungsmotiv für den Angriff der Caspase-6 dient. In einigen weiteren untersuchten Zelllinien wie Mono Mac 6 (MM6), RBL-1, PMNL oder HeLa, die nicht den B-Lymphozyten angehören, konnte die 5-LO-Spaltung weder durch das Passagieren von Zellen noch durch die Behandlung mit diversen proapoptotischen Agentien ausgelöst werden. Laut Ergebnissen aus in vitro-Untersuchungen scheinen 5-LO-positive HeLa- bzw. MM6-Zellen einen Faktor zu exprimieren, der die 5-LO direkt oder indirekt vor dem Angriff der Caspase-6 und anschließender Prozessierung schützt. Die in den BL41-E95-A-Zellen beobachtete Aktivierung der Caspasen mit anschließender Prozessierung der 5-LO lässt sich durch zwei Pflanzeninhaltsstoffe supprimieren, das Hyperforin (HP) aus Johanniskraut-Extrakten und das Myrtucommulon (MC) aus Myrte-Blättern. Beide Verbindungen scheinen in B-Lymphozyten zu einer Hemmung der Caspasen-Aktivierung zu führen. Nichtsdestotrotz führt die Behandlung der B-Lymphozyten mit HP bzw. MC zu einem apoptotischen Tod der Zellen. Offensichtlich wird dabei ein (unbekannter) einzigartiger Mechanismus der Apoptose-Induktion ausgelöst. In der vorliegenden Arbeit konnte zum ersten Mal eine potente Apoptose-induzierende Wirkung des natürlich vorkommenden Myrtucommulons auf Krebszelllinien gezeigt werden. In allen getesteten Krebszelllinien führte Myrtucommulon zum Zelltod, wobei die HL-60-Zellen mit einem IC50-Wert von 3,26 ± 0,51 µM MC am sensitivsten gegenüber MC-Einfluss waren. Zusätzlich konnte in HL-60- und MM6-Zellen nach MC-Behandlung neben einer erhöhten Caspasen-Aktivität und PARP-Spaltung ein signifikanter DNA-Abbau detektiert werden. Von besonderer Bedeutung ist die Tatsache, dass die zytotoxische MC-Wirkung eine bemerkenswerte Selektivität für entartete Zelllinien zu besitzen scheint und gegenüber nicht-transfizierten Zellen minimal ist.5-Lipoxygenase is the key enzyme in the synthesis of leukotriens (LT), bioactive metabolits of the arachidonic acid (AA). 5-LO protein is mainly expressed in myeloid cells, like granulocytes, monocytes, B-lymphocytes. The 5-LO undergoes proteolytic cleavage in the human B-lymphocytic cell line BL41-E95-A to a 62 kDa fragment. The degradation of 5-LO correlates with increased activity of caspase-8 and -6 and can be prevented by the protein synthesis inhibitor cycloheximide. Isolated recombinant human 5-LO is cleaved after the Asp170 incide the recognition motiv IQFD to a cleavage product of 58 kDa. No 5-LO cleavage can be observed in transfected HeLa or in MM6 cells after stimulation with caspase-activating apoptotic stimuli like TNF alpha, cycloheximide or daunorubicin, or in HeLa cells coexpressing 5-LO, caspase-6 and/or caspase-8. In contrast, processing of the typical caspase substrate PARP as well as of cytosolic phospholipase A2 (cPLA2) can be detected in 5-LO positive HeLa cells after stimulation with apoptotic agents. It appears that an unknown factor in 5-LO-positive HeLa and MM6 cells protects 5-LO against proteolysis by caspase-6 whereas other caspase substrates remain unaffected

The nature, extent and age of the lower crust and underlying subcontinental lithospheric mantle (SCLM) beneath the Siberian Craton (Russia)

Empirical thesis."Australian Research Council Centre of Excellence for Core and Crust Fluid Systems (CCFS) and GEMOC Department of Earth and Planetary Sciences, Macquarie University, Sydney, Australia" -- at foot of title.Bibliography: pages 295-316.Chapter 1. Introduction -- Chapter 2. The Siberian Craton : literature overview -- Chapter 3. Samples, analytical methods and techniques -- 4. The Magan terrane -- 5. The Markha terrane -- 6. The Daldyn terrane -- 7. The Khapchan terrane -- 8. The Birekta terrane -- 9. Discussion -- Chapter 10. Conclusions -- References -- Appendices.The lower continental crust and subcontinental lithospheric mantle (SCLM) are very interesting and important part of the Earth planet that has influenced significantly processes, which are continually altering surface of the Earth. Knowledge of the nature, structure and evolution of these layers may lead to understanding of the deep Earth’s processes.The Siberian craton is one of a few regions where samples of lower crust and underlying subcontinental lithospheric mantle can be found. Many geological and geophysical studies were carried out in this region but the role of crust and SCLM in the Earth’s evolution is not completely understood yet. Modern analytical techniques allow solving problems that were outstanding up to now. For example, isotopic and trace-element studies on zircon can specify not only the age of rock but also the source of melts and thus identify different stages in the craton evolution.The Siberian craton has experienced widespread Phanerozoic kimberlite magmatism. Besides diamonds, kimberlites also carry up a unique set of xenogenic material (xenoliths and xenocrysts) derived from depths of the lithosphere-asthenosphere transition zone (230-250 km) to the upper crust. Thus, kimberlitic pipes are natural drill holes, supplying with upper-mantle and crustal xenoliths and allowing us to investigate and reconstruct the structure of the deeper sections of the lithosphere.More than 1000 zircon grains from five terranes (Magan, Markha, Daldyn, Khapchan and Birekta) of the Siberian craton were analysed for U-Pb ages, trace-element characteristics, hafnium and oxygen isotopes composition. These data were supplemented by Re-Os isotope information collected on sulphide inclusion in silicate minerals from the mantle xenoliths. This combined isotopic information show that there were several events that involved both reworking of older crust and some addition of mantle-derived material. Moreover, integration of U-Pb age and Hf-isotopic data for zircons from different terranes indicates that the main magmatic-tectonic events are coeval for all studied terranes. For kimberlitic fields located within the Magan and Markha terranes the results of U-Pb and model ages show a good agreement with previous data. However, the ages from the eastern part of the craton (Khapchanand Birekta terranes) are much older than those reported previously. This implies that the amalgamation of the Siberian craton happened much earlier than it was previously suggested,which might have a significant impact on the diamond-bearing potential of the eastern part of the craton.Mode of access: World wide web1 online resource (405 pages) illustrations (some colour), map

Phosphatidylinositol Stabilizes Fluid-Phase Liposomes Loaded with a Melphalan Lipophilic Prodrug

Previously, a liposomal formulation of a chemotherapeutic agent melphalan (Mlph) incorporated in a fluid lipid bilayer of natural phospholipids in the form of dioleoylglyceride ester (MlphDG) was developed and the antitumor effect was confirmed in mouse models. The formulation composed of egg phosphatidylcholine (ePC), soybean phosphatidylinositol (PI), and MlphDG (8:1:1, by mol) showed stability in human serum for at least 4–5 h. On the contrary, replacing PI with pegylation of the liposomes, promoted fast dissociation of the components from the bilayer. In this work, interactions of MlphDG-liposomes with the most abundant plasma protein—albumin—in function of the presence of PI in the formulation were explored using Fourier transform infrared spectroscopy. The release of MlphDG from the liposomes was studied by asymmetrical flow field-flow fractionation (AF4) using micelles formed by a polyethylene glycol conjugate with phosphatidylethanolamine to mimic the physiological lipid sink like lipoproteins. Our results show that PI actually protects the membrane of MlphDG-liposomes from the protein penetration, presumably due to pairing between the positively charged MlphDG and negatively charged PI, which compensates for the heterogeneity of the lipid bilayer. The AF4 technique also evidences high stability of the formulation as a drug carrier

Spectroscopy Study of Albumin Interaction with Negatively Charged Liposome Membranes: Mutual Structural Effects of the Protein and the Bilayers

Liposomes as drug carriers are usually injected into the systemic circulation where they are instantly exposed to plasma proteins. Liposome–protein interactions can affect both the stability of liposomes and the conformation of the associated protein leading to the altered biodistribution of the carrier. In this work, mutual effects of albumin and liposomal membrane in the course of the protein’s adsorption were examined in terms of quantity of bound protein, its structure, liposome membrane permeability, and changes in physicochemical characteristics of the liposomes. Fluorescence spectroscopy methods and Fourier transform infrared spectroscopy (ATR-FTIR), which provides information about specific groups in lipids involved in interaction with the protein, were used to monitor adsorption of albumin with liposomes based on egg phosphatidylcholine with various additives of negatively charged lipidic components, such as phosphatidylinositol, ganglioside GM1, or the acidic lipopeptide. Less than a dozen of the protein molecules were tightly bound to a liposome independently of bilayer composition, yet they had a detectable impact on the bilayer. Albumin conformational changes during adsorption were partially related to bilayer microhydrophobicity. Ganglioside GM1 showed preferable features for evading undesirable structural changes

Myrtucommulone from Myrtus communis induces apoptosis in cancer cells via the mitochondrial pathway involving caspase-9.

Myrtucommulone (MC) is a unique, nonprenylated acylphloroglucinol contained in the leaves of myrtle (Myrtus communis). Here, we addressed the potential of MC to induce apoptosis of cancer cells. MC potently induced cell death of different cancer cell lines (EC(50) 3-8 microM) with characteristics of apoptosis, visualized by the activation of caspase-3, -8 and -9, cleavage of poly(ADP-ribose)polymerase (PARP), release of nucleosomes into the cytosol, and DNA fragmentation. MC was much less cytotoxic for non-transformed human peripheral blood mononuclear cells (PBMC) or foreskin fibroblasts (EC(50) cell death = 20-50 microM), and MC up to 30 microM hardly caused processing of PARP, caspase-3, -8 and -9 in human PBMC. MC-induced apoptosis was mediated by the intrinsic rather than the extrinsic death pathway. Thus, MC caused loss of the mitochondrial membrane potential in MM6 cells and evoked release of cytochrome c from mitochondria. Interestingly, Jurkat cells deficient in caspase-9 were resistant to MC-induced cell death and no processing of PARP or caspase-8 was evident. In cell lines deficient in either CD95 (Fas, APO-1) signalling, FADD or caspase-8, MC was still able to potently induce cell death and PARP cleavage. Conclusively, MC induces apoptosis in cancer cell lines, with marginal cytotoxicity for non-transformed cells, via the mitochondrial cytochrome c/Apaf-1/caspase-9 pathway

Recurrent magmatic activity on a lithosphere-scale structure: Crystallization and deformation in kimberlitic zircons

Kimberlites are not only the most economically important source of diamonds; they also carry unique information encapsulated in rock fragments entrained as the magma traverses the whole thickness of the lithosphere. The Nurbinskaya pipe in the Siberian kimberlite province (Russia) is one of several intruded along the Vilyui Rift, a major terrane boundary. The pipe contains three populations of mantle-derived zircon xenocrysts: Archean (mean age 2709 ± 9 Ma), Devonian (mean age 371 ± 2.3 Ma), and a subset of grains with evidence of brittle deformation and rehealing, and a range of ages between 370 and 450 Ma. The Hf-isotope, O-isotope and trace-element signatures of the last group provide a link between the Archean and Devonian events, indicating at least three episodes of magmatic activity and zircon crystallization in the lithosphere beneath the pipe. The emplacement of the Nurbinskaya pipe ca 370 Ma ago was only the youngest activity in a magma plumbing system that has been periodically reactivated over at least 2.7 billion years, controlled by the lithosphere-scale structure of the Vilyui Rift

Multidisciplinary Observations across an Eddy Dipole in the Interaction Zone between Subtropical and Subantarctic Waters in the Southwest Atlantic

Seawater properties in two intense rings in the South Atlantic are considered. One ring separated from the Brazil Current and the other from the Malvinas Current. The analysis is based on the CTD casts and SADCP measurements from the onboard velocity profiler. The optical properties, chemical parameters, methane concentration, and biological properties such as primary production, plankton, and fish were also analyzed. Analysis of strong differences between the eddies is supplemented by observations of whales and birds in the region

Multidisciplinary Observations across an Eddy Dipole in the Interaction Zone between Subtropical and Subantarctic Waters in the Southwest Atlantic

Seawater properties in two intense rings in the South Atlantic are considered. One ring separated from the Brazil Current and the other from the Malvinas Current. The analysis is based on the CTD casts and SADCP measurements from the onboard velocity profiler. The optical properties, chemical parameters, methane concentration, and biological properties such as primary production, plankton, and fish were also analyzed. Analysis of strong differences between the eddies is supplemented by observations of whales and birds in the region