Die Regulation erythrozytären Überlebens und suizidalen Zelltodes

The life span of erythrocytes is tightly regulated. Therefore, a mechanism is required to remove senescent or damaged erythrocytes without rupture of the cell membrane resulting in the release of hemoglobin which may impair kidney function. The mechanism of suicidal erythrocyte death is called eryptosis and shares similarities with apoptosis of nucleated cells such as exposure of phosphatidylserine at the cell surface, increase in cytosolic Ca2+ concentration, blebbing of the membrane, cell shrinkage and enzymatic degradation of the cytoskeletton. The cell shrinkage of eryptotic cells is mediated by a Ca2+-dependent K+ channel, the Gardos channel. Its activation by an increase in the intracellular Ca2+ concentration results in the efflux of K+, Cl- and osmotically obliged water. Phosphatidylserine-exposing erythrocytes are rapidly engulfed by macrophages equipped with phosphatidylserine receptors and degraded. Excessive eryptosis may lead to anemia, the pathological lack of erythrocytes. The present study was performed to elucidate mechanisms regulating erythrocyte survival and suicidal cell death. First, the functional significance of the Gardos channel for suicidal erythrocyte death and erythrocyte clearance was studied. Furthermore, the protective role of Gardos channels during exposure to hemolytic toxins was elucidated. Both issues were addressed by experiments performed in mice lacking the Ca2+-dependent K+ channel KCa3.1, the Gardos channel, and their wildtype littermates. Using patch-clamp recording, flow cytometry, in vitro hemolysis and a mouse sepsis model, it is shown that Gardos channel activity and Gardos effect delay hemolysis of injured erythrocytes and, thus, prevent the disastrous filtration of released hemoglobin into the renal tubular system. In a further series of experiments, the role of the NO/cGMP pathway, a powerful regulator of the life span of a variety of cells, for erythrocyte survival is investigated. Flow cytometry, Western Blotting, hematological counts, and MRI imaging were used to illustrate by means of a cGKI-deficient mouse model that cGKI is a mediator of erythrocyte survival in vitro and in vivo. Moreover, the participation of the phosphoinositide-dependent kinase PDK1, a key element in the phosphoinositol-3-kinase signalling pathway, which is involved in the regulation of ion channels, transporters, cell volume and cell survival, in the regulation of suicdal erythrocyte death was studied. Experiments performed in hypomorphic mice with some 20% of normal PDK1 acitivity and their wildtype littermates revealed that PDK1 deficiency is associated with decreased Ca2+ entry into erythrocytes and thus with blunted eryptotic effects of oxidative stress, osmotic shock and chloride removal. Finally, the functional significance of host pathogen interactions for suicidal erythrocyte death was investigated. Using flow cytometry, it could be shown that peptidoglycan, a main component of the bacterial cell wall, is a potent stimulus of eryptosis and thereby impairs erythrocyte survival. Peptidoglycan-induced eryptosis may therefore, at least in part, account for anemia observed in patients with bacterial infections.Die Lebensdauer von Erythrozyten ist genau reguliert. Aus diesem Grund ist ein Mechanismus erforderlich, der es erlaubt, alte oder geschädigte Erythrozyten abzubauen, ohne daß die Zellmembran reißt und Hämoglobin freigesetzt wird, was zu akutem Nierenversagen führen könnte. Ein solcher Mechanismus ist der suizidale Erythrozytentod, der Eryptose genannt wird und Ähnlichkeiten zur Apoptose kernhaltiger Zellen aufweist wie zum Beispiel die Externalisierung von Phosphatidylserin auf der Zelloberfläche, die Zunahme der intrazellulären Calciumkonzentration, das Abknopsen von Membranteilen, Zellschrumpfung sowie der enzymatische Abbau des Zytoskeletts. Die Zellschrumpfung eryptotischer Zellen wird durch einen Ca2+-abhängigen K+-Kanal, den Gardos-Kanal, vermittelt. Dessen Aktivierung durch die Erhöhung der intrazellulären Calciumkonzentration führt zum Verlust von K+, Cl- und osmotisch folgendem Wasser. Phosphatidylserin-exponierende Erythrozyten werden zügig von Makrophagen, die über Phosphatidylserinrezeptoren verfügen, phagozytiert und abgebaut. Exzessiv gesteigerte Eryptose kann zu Anämie, dem krankhaften Mangel an roten Blutkörperchen, führen. Ziel der vorliegenden Arbeit war es, Mechanismen aufzuklären, die erythrozytäres Überleben sowie suidzidales Sterben steuern. Zunächst wurde die funktionelle Bedeutung des Gardos-Kanals für den suizidalen Erythrozytentod und die Klärung der roten Blutkörperchen aus dem Blut untersucht. Ferner wurde die protektive Wirkung der Gardos-Kanäle bei Einwirkung hämolytischer Toxine studiert. Beide Fragestellungen wurden durch Experimente an Mäusen, die den Ca2+-abhängigen K+-Kanal KCa3.1 nicht exprimierten, und ihren Wildtyp-Geschwistern angegangen. Durch Messungen mit der Membranfleckklemme, mit dem Durchflußzytometer, durch in vitro Hämolyse und mittels eines Maus-Sepsis-Modells wurde gezeigt, daß die Gardos-Kanal-Aktivität und der Gardos-Effekt die Hämolyse geschädigter Erythrozyten verzögern und dadurch die potentiell schädliche Filtration des hämolytisch freigesetzten Hämoglobins in das Nierentubulssystem unterbinden. In weiteren Experimenten wurde die Rolle des NO/cGMP-Signalweges, eines bedeutsamen Regulators der Lebensdauer verschiedener Zellen, für das erythrozytäre Überleben untersucht. Mittels Durchflußzytometrie, Western Blotting, Blutbildern sowie Kernspintomographie, wurde an einem cGKI-defizienten Maus-Modell gezeigt, daß die cGKI ein Mediator erythrozytären Überlebens in vitro und in vivo ist. Desweiteren wurde die Beteiligung der Phosphoinositid-abhängigen-Kinase PDK1, eines Schlüsselenzyms des Phosphoinositol-3-kinase-Signalweges, der bei der Regulation von Ionenkanälen, Transportern, des Zellvolumens und –überlebens mitwirkt, an der Steuerung suizidalen Erythrozytentodes analysiert. Experimente an hypomorphen Mäusen mit lediglich 20%-iger PDK1-Aktivität und ihren Wildtyp-Geschwistern ergaben, daß PDK1-Defizienz mit vermindertem Calciumeinstrom in Erythrozyten und daher mit abgeschächten eryptotischen Effekten von oxidativem Streß, osmotischem Schock und der Entfernung extrazellulären Chlorids verbunden ist. Schließlich wurde die funktionelle Signifikanz von Wirt-Erregerbeziehungen für suizidalen Erythrozytentod untersucht. Durchflußzytometrisch konnte gezeigt werden, daß Peptidoglykan, ein wesentlicher Bestandteil bakterieller Zellwände, ein potenter Auslöser von Eryptose ist und dadurch erythrozytäres Überleben beeinträchtigt. Zumindest theoretisch könnte daher Peptidoglykan-induzierte Eryptose zur Anämie von Patienten mit bakteriellen Infektionen beitragen

Constant diversification rates of endemic gastropods in ancient Lake Ohrid: ecosystem resilience likely buffers environmental fluctuations

Ancient lakes represent key ecosystems for endemic freshwater species. This high endemic biodiversity has been shown to be mainly the result of intra-lacustrine diversification. Whereas the principle role of this mode of diversification is generally acknowledged, actual diversification rates in ancient lakes remain little understood. At least four types are conceivable. Diversification rates may be constant over time, they may fluctuate, rates may be higher in the initial phase of diversification, or there may be a pronounced lag phase between colonization and subsequent diversification. As understanding the tempo of diversification in ancient lake environments may help reveal the underlying processes that drive speciation and extinction, we here use the Balkan Lake Ohrid as a model system and the largest species flock in the lake, the non-pyrgulinid Hydrobiidae, as a model taxon to study changes in diversification rates over time together with the respective drivers. Based on phylogenetic, molecular-clock, lineage-through-time plot, and diversification-rate analyses we found that this potentially monophyletic group is comparatively old and that it most likely evolved with a constant diversification rate. Preliminary data of the SCOPSCO (Scientific Collaboration On Past Speciation Conditions in Lake Ohrid) deep-drilling program do indicate signatures of severe environmental/climatic perturbations in Lake Ohrid. However, so far there is no evidence for the occurrence of catastrophic environmental events. We therefore propose that the constant diversification rate observed in endemic gastropods has been caused by two factors: (i) a potential lack of catastrophic environmental events in Lake Ohrid and/or (ii) a probably high ecosystem resilience, buffering environmental changes. Parameters potentially contributing to the lake´s high ecosystem resilience are its distinct bathymetry, ongoing tectonic activities, and karst hydrology. The current study not only contributes to one of the overall goals of the SCOPSCO deep-drilling program – inferring the driving forces for biotic evolution in Lake Ohrid. It might also enhance our understanding of how ecosystem resilience, in general, may promote relatively constant diversification rates in isolated ecosystems. However, we encourage future studies testing hypotheses about the lack of catastrophic events in Lake Ohrid. These studies should be based on high-resolution data for the entire geological history of the lake, and they should potentially involve information from the sediment fossil record, not only for gastropods but also for other groups with a high share of endemic taxa

Beneficial effect of aurothiomalate on murine malaria

<p>Abstract</p> <p>Background</p> <p>Premature death of <it>Plasmodium</it>-infected erythrocytes is considered to favourably influence the clinical course of malaria. Aurothiomalate has previously been shown to trigger erythrocyte death or eryptosis, which is characterized by cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing cells are rapidly cleared from circulating blood. The present study thus tested whether sodium aurothiomalate influences the intraerythrocytic parasite development <it>in vitro </it>and the clinical course of murine malaria <it>in vivo</it>.</p> <p>Methods</p> <p>Human erythrocytes were infected with <it>Plasmodium falciparum </it>BinH <it>in vitro </it>and mice were infected (intraperitoneal injection of 1 × 10⁶parasitized murine erythrocytes) with <it>Plasmodium berghei </it>ANKA <it>in vivo</it>.</p> <p>Results</p> <p>Exposure to aurothiomalate significantly decreased the <it>in vitro </it>parasitemia of <it>P. falciparum</it>-infected human erythrocytes without influencing the intraerythrocytic DNA/RNA content. Administration of sodium aurothiomalate <it>in vivo </it>(daily 10 mg/kg b.w. s.c. from the 8^thday of infection) enhanced the percentage of phosphatidylserine-exposing infected and noninfected erythrocytes in blood. All nontreated mice died within 30 days of infection. Aurothiomalate-treatment delayed the lethal course of malaria leading to survival of more than 50% of the mice 30 days after infection.</p> <p>Conclusions</p> <p>Sodium aurothiomalate influences the survival of <it>Plasmodium berghei</it>-infected mice, an effect only partially explained by stimulation of eryptosis.</p