Browsing by Subject "Reaktive Sauerstoffspezies"

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    Die Bedeutung von Aquaporinen und ihren Interaktionspartnern für die Zelltodregulation in Pflanzen
    (2011) Hoch, Tanja; Pfitzner, Artur J. P.
    Programmed cell death (PCD, apoptosis) is an induced cell suicide process that plays an important role during the differentiation and pathogen defense responses of plants and animals. BHRF1 (?BamHI fragment H rightward open reading frame no. 1?) is a cell-death modulating protein of the Epstein-Barr virus (EBV), a human lymphotrophic herpes virus. The expression of BHRF1 in transgenic plants led to the formation of necrotic lesions. Further experiments showed that BHRF1 associated necrotic lesions are caused due to stress, senescence and pathogen defense responses. Yeast-two-hybrid-screening of a tobacco cDNA library identified two different aquaporins as partners for interacting with BHRF1. Aquaporins were identified as water channels/carriers within red blood cells, but are also present in all other organisms. Over the last years, more information was gathered indicating that, apart from transporting water, aquaporins had other functional activities. E. g. Henzler and Steudle (2000) demonstrated that aquaporins can act as hydrogen peroxide channels in the algae Chara corallina. Furthermore, publications by Bienert et al. (2006), indicating that aquaporins in plants as well as in animals are also able to transport H2O2. Hydrogen peroxide and other reactive oxygen species (ROS) have long been recognized as important signal molecules during the pathogen defense response in plants, therefore establishing a logical connection between cell death and aquaporins for the first time. It was assumed that the aquaporins NtPIP2.2a, NtPIP2.2b und NtTIP1.1a identified during the yeast-two-hybrid-screen can act as H2O2 channels. In further experiments it could indeed be established that these aquaporins have the ability to transport H2O2 in yeast cells. Yeasts expressing aquaporins could be influenced in their H2O2 sensitivity by the expression of BHRF1. BHRF1 without transmembrane domain (BHRF1deltaTMwt) led to an enhanced H2O2 sensitivity and also to an increase in cell death. In addition, the transient expression of aquaporin could induce necrotic lesions and cell death in Nicotiana benthamiana. Deletion experiments identified a common binding domain for interacting with BHRF1 in these aquaporins. This binding domain consists of the conserved region containing the first NPA motive (?loop? B) that is also half of one pore. Further studies showed that BHRF1 interacts with all kinds of different aquaporins from plants, animals (rAQP8) and humans (hAQP1). BHRF1 most likely binds with the alpha3 helix to the highly conserved NPA region of aquaporins. A cellular protein showing sequence homology to M20 proteases and aminoacylases was isolated when looking for interaction partners of aquaporins in plants. Like BHRF1, this protein binds to the conserved NPA region of the aquaporins. Although the cellular substrate for this protein has to be found yet, an interesting observation was made. Co-expression of the isolated aminoacylase with NtTIP1.1a or NtPIP2.2b in Nicotiana benthamiana led to the inhibition of cell death induced by these aquaporins.
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    Mechanismen der Resistenzinduktion nach Blattbehandlungen mit Phosphaten
    (2002) Orober, Miroslav; Buchenauer, Heinrich
    In this study the induction of resistance against plant diseases following foliar application of phosphates was investigated. The early biochemical responses of the resistance activation by phosphates have been compared with other forms of induction of SAR such as the biotic induction with pathogens and treatment with synthetic plant defense activators. In cucumber plants foliar applications of phosphates lead to increased local and systemic acquired resistance against fungal pathogens such as Colletotrichum lagenarium, Sphaerotheca fuliginea and Pseudoperonospora cubensis. In tobacco foliar phosphate application enhanced resistance against TMV. For successful induction of SAR the occurrence of chlorotic/necrotic lesions on the phosphate treated inducer leaves was necessary. These reactions were accompanied by the occurrence of localized cell death, which was preceded by the generation of reactive oxygen species such as superoxide anions and hydrogen peroxide. Enhanced lipid peroxidation was observed in the treated leaves. Local phosphate treatments induced an increase of the concentrations of free and bound salicylic acid in the treated and distal leaves. Experiments with transgenic nahG-tobacco plants showed that the expression of SAR by phosphate treatments and TNV-inoculation was strictly dependent on the accumulation of salicylic acid. The activities of characteristic defense-related enzymes like peroxidases and polyphenoloxidases were highly increased in treated and in the distal leaves, respectively. In this study it could be shown that treatments with necrotizing chemicals such as phosphates cause similar cellular reactions as observed after biotic induction with pathogens which resulted in expression of SAR. Therefore it can be assumed that foliar phosphate treatments imitate the biotic induction of systemic acquired resistance.
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    Role of reactive oxygen species in anti-cancer treatment: Investigations in 2-methoxyestradiol chemotherapy and 5-aminolevulinic acid based photodynamic therapy combined with hyperthermia
    (2003) Lambert, Christine; Frank, Jürgen
    The thesis deals with two different ROS-generating anti-cancer treatments: chemotherapy with the endogenous estrogen metabolite 2-methoxyestradiol and 5-aminolevulinic acid based photodynamic therapy. Both treatments were investigated with the rat DS-sarcoma model, which can be used in vitro and in vivo. It the first part, it could be shown that 2-methoxyestradiol induces apoptosis in DS-sarcoma cells. Translocation of the pro-apoptotic protein Bax to the mitochondria was identified as initial apoptotic event, followed by a decrease in mitochondrial transmembrane potential and the release of AIF out of the mitochondria. In addition, upregulation of FasL and TNFalpha by 2-ME, two death receptor ligands, was observed. Although, 2-ME administration resulted in activation of caspases, pan caspase inhibitor Z-VAD-FMK could not block 2-ME induced apoptotic cell death pointing to a caspase-independent mechanism. Furthermore, an increase in formation of reactive oxygen species was observed after 2-ME treatment. However, supplementation with different antioxidants could not decrease the toxic effect of 2-ME. This finding may indicate, that reactive oxygen species are not involved in apoptosis induction, rather they are a consequence of mitochondrial damage. In vitro and in vivo combination of 2-ME with another ROS-generating treatment resulted in a synergistic anti-tumour effect. In the second part of the thesis anti-tumour effects of 5-aminolevulinic acid based photodynamic therapy combined with simultaneous hyperthermia was investigated. Analysis of apoptosis associated nuclear changes clearly demonstrated the high efficiency of this treatment regime. Formation of reactive compounds (e.g. ROS, nitrogen monoxide, peroxynitrite) which is mainly responsible for toxicity of PDT, could be assessed in the shape of massive protein nitrosylation in tumours treated with PDT alone or the combined treatment. Detection of decreased amounts of heat shock proteins (HSP70 and HO-1) which protect tumour cells against damaging influences, lowered glutathione levels and reduced MMP-activity indicate an increase in degradation of proteins. This phenomenon may be caused by excessive generation of ROS. Taken together, the presented studies could demonstrate the high benefit of combining 2-ME resp. ALA-PDT with hyperthermia (or other ROS-generating therapies), which make them interesting candidates for future clinical applications.