Institut für Biologie

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Changes in the concentration of particular hormones and carbohydrates in apple shoots after "bending" respectively chemical treatments and relationship to the flower induction process
(2005) Boonplod, Nopporn; Bangerth, Fritz
SUMMARY Apples are cultivated commercially throughout the temperate zone. A regular production however does not seem possible because of irregular yields from year to year. Main causes for this are the so called "alternate bearing" behavior which is the result of profuse flowering in one year but few or no flowers in the following year. It is reported that too vigorously growing shoots are part of the reasons for alternate bearing in apple trees. Applications of chemicals or conventional cultural practices, such as bending shoots have been widely used to restrict shoot growth and promote flower induction. However, the physiological mode of action of these methods in FI is still unknown. Phytohormones are thought to be involved in the process of flower induction (FI). In the above experiments, we investigated changes in endogenous hormones, starch and sugar contents after bending upright shoots into a horizontal position and spraying apple trees with the growth regulators Alar plus Ethrel to improve FI. The experiments were carried out during the years 2001 to 2003 at the Experiment Station, of the University of Hohenheim, Germany, whereby the apple cvs. ?Golden Delicious?, ?Boskoop?, ?Elstar? and ?Idared? were used. The apical part of growing shoots and non-growing bourse shoots, beside bark, wood and shoot diffusates were collected. Plant samples were frozen immediately in liquid nitrogen and freeze dried. Phosphate buffer 0.1M, pH 6.2 was used for collecting auxin in the shoot diffusates. All samples were stored at ?20C until extraction and purified, identified and quantified by Radio Immuno Assay (RIA). The results revealed, in general, that shoot bending and spraying with Alar plus Ethrel changed the endogenous hormone concentrations in the apical part of shoots, as well as in wood, bark and shoot exudates of apple trees. The ?Golden Delicious? cultivar and vigorously growing shoots showed clearer tendencies of hormonal changes than the other cvs. and non-growing bourse shoots. Cytokinin concentrations in the apical part of shoots, and in wood and bark increased after both treatments. Contrary to that, GAs and IAA concentrations in the apical part of shoots and in shoot exudates showed the opposite results. Both treatments had no effect on the concentration of ABA. Ethylene production in shoot tips was considerably stimulated by the combined treatment of Ethrel plus Alar probably due to Ethrel being a "synthetic precursor" of ethylene. Considerable variation existed in the mentioned hormonal changes in respect to the year of examination and the cv. under investigation. Time of treatments and in particular climatic conditions were probably the most influential variables. In spite of all this and on the basis of the above results the conclusion can be drawn that higher concentrations of cytokinins and lower concentrations of gibberellins and auxin are favorable for FI. Spraying with Alar plus Ethrel and bending of shoots seemed to decrease the reducing-sugars, as well as sucrose and starch concentrations in growing shoots and their leaves. In non-growing shoots, spraying seemed to reduce starch but to increase reducing-sugars and sucrose concentrations. A correlation between changes in carbohydrate contents (reducing sugar, sucrose and starch) caused by the spraying treatments and FI does not seem to exist. All the observed changes in the carbohydrate concentrations caused by spraying treatments were not particular impressive and did not really support the often published claim that the effect of spraying growth regulators, bending shoots or other cultural practices may mediate their stimulatory effect on FI via a change in carbohydrates. In contrast to that the above observed experimental results rather suggest that hormones are more effectively involved in the flower induction process of fruit trees.
Functional characterization of the COOH-terminal kinase activity of the TBP-associated factor TAF1
(2006) Maile, Tobias; Sauer, Frank
Activation of eukaryotic transcription involves an orchestrated interplay between transcription factors and the general RNA polymerase II (Pol II) transcription machinery (GTM), which consists of Pol II and general transcription factors (GTFs). The GTF TFIID consists of the TATA-box binding protein (TBP) and several TBP-associated factors (TAFs). The binding of TFIID to promoters can nucleate transcription. TAF1 is the largest subunit of TFIID and plays a central role within the nucleating function of TFIID in transcription. TAF1 mediates the binding of TFIID to promoters and interacts with enhancer-bound transcription factors and several GTFs. Additionally, TAF1 contains four enzymatic activities that are essential for viability of eukaryotes and mediate posttranslational modification of GTFs and histones. TAF1 is a bipartite protein kinase and contains an NH2-terminal kinase domain (NTK) and a COOH-terminal kinase domain (CTK). A previous study demonstrated that the CTK phosphorylates serine-residue 33 in histone H2B (H2BS33). However, the role of TAF1-mediated phosphorylation in transcription regulation remained unknown. In this study, the functional importance of H2BS33 phosphorylation (H2BS33P) by TAF1 was investigated by using a combination of biochemical and in vivo assays. In vitro kinase assays uncovered the two essential kinase motifs in TAF1CTK, the ATP-binding motif and the serine/threonine-specific catalytic motif, and indicate that the TAF1 CTK has intrinsic kinase-activity. Western blot analysis using an antibody to H2BS33P revealed that H2BS33 is phosphorylated in Drosophila. RNA-interference (RNAi) assays, designed to attenuate TAF1 expression (TAF1RNAi), revealed that TAF1 is a major kinase for H2BS33 in Drosophila Schneider cells. Flow-cytometry analysis of TAF1RNAi cells indicated that loss of TAF1 expression results in cell cycle arrest in G2/M-phase. Screening the transcription of cell cycle genes in TAF1RNAi cells by using reverse-transcriptase-PCR demonstrated that the transcription of the cell cycle gene string (stg) is reduced in the absence of TAF1. Chromatin immunoprecipitation assays (XChIP) indicate that H2BS33P is detectable at the transcriptionally active stg promoter but not at the silent stg promoter in TAF1RNAi cells. These results demonstrate that phosphorylation of H2BS33 is involved in stg transcription. XChIP-assays using chromatin prepared from Drosophila embryos, which express a mutant TAF1 lacking the CTK, revealed that CTK-mediated phosphorylation of H2BS33 plays an essential role in the activation of transcription of the Drosophila segmentation gene giant. In vitro kinase assays demonstrate that Bdf1 and Bdf2, the yeast homologues of the TAF1CTK, phosphorylate histones suggesting that the kinase activity of the TAF1CTK is phylogenetically conserved. The results of this work demonstrate that TAF1CTK is a major histone kinase of H2BS33 and that TAF1-mediated phosphorylation of H2BS33 plays an essential role in the transcription events during cell cycle progression and development.
Funktionen charakteristischer Sequenzmotive endogener und toxischer mitochondrialer Proteine
(2006) Papatheodorou, Panagiotis; Rassow, Joachim
In the course of their biogenesis, mitochondria take up nuclear encoded proteins from the cytosol continuously. Protein import at the mitochondrial outer membrane is mediated by TOM proteins and by TIM proteins at the inner membrane, respectively. Now and then, toxical proteins released by pathogenic bacteria to infected tissue can also reach mitochondria. The present dissertation provides new findings on the role of characteristical sequence motifs that can be identified in endogenous and toxical mitochondrial proteins. In an extensive project the importance of sequence motifs from mitochondrial metabolite carrier proteins in their biogenesis and function was investigated in more detail. It could be shown, that the positively charged presequence of the citrate carrier from Rattus norvegicus is not involved in mitochondrial targeting but rather serves as an internal chaperone. A conserved sequence motif, PX(D/E)XX(R/K), the Carrier Signature, which can be found in all mitochondrial carrier proteins, does also not represent a mitochondrial targeting signal, as could be proven by using the dicarboxylate carrier from Saccharomyces cerevisiae as a model protein. Even the translocation across the outer membrane, the insertion into the inner membrane and the following dimerization of the dicarboxylate carrier are processes occuring independently of the Carrier Signature. Instead, it was discovered, that the Carrier Signature is primarily necessary for the function of metabolite carrier proteins in the inner membrane. In another project it could be shown for the Map toxin from enteropathogenic Escherichia coli strains (EPEC), that it is directed to the mitochondrial matrix, mediated by its typical N-terminal presequence and by the TOM and TIM complexes, respectively. The Map toxin leads then to the fragmentation of the mitochondrial network independent of the mitochondrial fission machinery and to the loss of the mitochondrial membrane potential. Moreover, it could be proven, that an internal conserved sequence motif, WXXXE, is essential for cytotoxicity of the Map toxin in the cytosol and for fission of mitochondria. A lysine residue within the WXXXE sequence serves probably as a locus of sumoylation. The investigations show, that mechanisms of intracellular protein transport are not only important for the biogenesis of mitochondria, but can also be relevant for pathological processes.
Determination of Laterality in the Rabbit Embryo: Studies on Ciliation and Asymmetric Signal Transfer
(2007) Feistel, Kerstin; Blum, Martin
The midline of the vertebrate embryo plays a pivotal role in the regulation of left-right (LR) asymmetry. In mammals recent interest has focused on a structure situated at the caudal part of the notochord, the posterior notochord (PNC), which is homologous to Kupffer?s vesicle (KV) in fish and the gastrocoel roof plate (GRP) in frog. Despite highly diverging embryonic architecture, the PNC/KV/GRP is the site where motile monocilia set up a directional fluid flow, an event indispensable for the generation of LR asymmetry. Signals created at the PNC/KV/GRP need to be transferred to the periphery of the embryo, where they initiate the left-specifying program in the left lateral plate mesoderm (LPM). In this study morphogenesis and ciliogenesis of the notochordal plate as well as the signaling processes between midline and LPM were studied in the rabbit embryo. Rabbit development progresses through a flat blastodisc phase and represents the typical mode of mammalian embryogenesis. Transcription of ciliary marker genes, the first sign of beginning ciliogenesis, initiated in Hensen?s node and persisted in the nascent notochord. Cilia emerged on cells leaving Hensen?s node anteriorly to form the notochordal plate. Cilia lengthened to about 5µm and polarized from an initially central position to the posterior pole of cells. Electron microscopic analysis revealed 9+0 and 9+2 cilia and a novel 9+4 axoneme intermingled in a salt-and-pepper-like fashion. These data showed that the ciliogenic gene program essential for laterality determination is conserved at the midline of the rabbit embryo. The present study also provided evidence that initiation as well as repression of the Nodal cascade crucially depended on communication between midline and lateral plate (LP). Separation of LP tissue from the midline before, during and after the 2 somite stage demonstrated that signals from the PNC induced and maintained the competence of LPM to express Nodal. Signals from the midline were necessary after the 2 somite stage to maintain a right-sided identity, i.e. absence of Nodal expression. Gap-junction-dependent intercellular communication (GJC) was shown to play a central role in this process. Previously, GJC had been involved in LR axis determination in cleavage stage frog embryos and early blastodisc stages in chick. This study for the first time demonstrates the role of GJC in mammalian embryos. GJs regulate the signaling between midline and periphery: permeable gap junctions were required specifically at the 2 somite stage to repress Nodal induction in the right LPM, whereas closed GJs were a prerequisite for Nodal signaling on the left side. Establishment of the right-sided fate depended on FGF8, the signaling of which was regulated by the opening status of GJs. A 3-step model is proposed for symmetry breakage and induction of the LR signaling cascade in vertebrates: (1) Nodal protein synthesized at the lateral edges of the PNC diffuses bilaterally and confers competence for the induction of the Nodal cascade to the LPM, (2) at the same time the left-specific cascade is actively repressed by action of the GJC/FGF8 module, and (3) following the onset of leftward flow at the PNC repression gets released specifically on the left side at the 2 somite stage, presumably by transient inhibition of GJC. This model not only is consistent with the presented data, but also with published work in other model organisms.
Epidemiologische Aspekte der Falschen Mehltauinfektion durch Plasmopara viticola an Vitis
(2007) Keil, Sven; Spring, Otmar
The obligate biotrophic oomycete Plasmopara viticola (Berk. & Curt.) Berl. & de Toni causes downy mildew on grapevine. Plasmopara viticola is one of the economically most important pathogens in viticulture, with severe losses in yield of up to 70%. Existing prognosis models for plant protection in viticulture only allow yes/no statements on possible infection events in the vineyard. On the basis of these models, the severity of infections remain uncertain, although this represents a crucial point for an efficient application of fungicides. At low infestation severity, the application of protective fungicides at the end of the incubation period usually is sufficient. The low level of infestation can be tolerated and only further propagation of the pathogen must be prevented. In contrast, at high infestation severity curative fungicides have to be applied as soon as possible, because otherwise too much host tissue would be destroyed. Based on epidemiological studies and field experiments a prognostic concept has been designed, which enables the user to evaluate the relevance of infection events of grapevine downy mildew. This work has been carried out in the context of the research project ?Optimierung der Peronospora-Bekämpfung im Rebschutz auf der Basis eines erweiterten Prognosemodells (Forschungsvorhaben des Bundesministeriums für Ernährung, Landwirtschaft und Verbraucherschutz Nr. 514-33.54/01HS048)?. The developed concept was then integrated into the existing prognosis model and should support both consultants and winegrowers in using plant protection agents only in case of an expected increase of infestation frequency and severity. In this way, an effective and more economical use of fungicides is possible, which contributes to economic savings and reduces pesticide contamination of the environment. In the present study, aspects of sporangiogenesis, infestation of host tissue and hibernation, spreading of sporangia, interaction between vine leaves and sporangia as well as the climatic conditions during infection were analysed and evaluated with respect to the impact on epidemiology. The results improve existing literature data and deliver new insight to the epidemiology and biology of Plasmopara viticola.
Funktionelle Untersuchung der Sensorkinase KdpD von Escherichia coli mit Hilfe verschiedener KdpD-Deletionsmutanten
(2007) Rothenbücher, Marina; Kuhn, Andreas
The high affinity K+ transport system KdpFABC is one of several uptake systems that accumulate K+ in Escherichia coli. Expression of the kdpFABC operon is under control of the regulatory proteins KdpD and KdpE, which constitute a typical sensor kinase/response regulator system. KdpD is an integral protein of the cytoplasmic membrane. The N-terminal domain, the 4 helices and 200 amino acids of the cytoplasmic C-terminal domain are accredited to be involved in the signal input function. Surprisingly, a mutant (KdpD-C) lacking the N-terminal domain, helix 1 and 2 is a functional K+ sensor, which is able to detect the changes in K+ concentration in the medium. To investigate which parts of the KdpD protein are essential for signal transduction, various truncated KdpD variants were constructed and analyzed. The results show that the fragment C499-894, which contains only the cytoplasmic C-terminal domain of KdpD, is able to recognise the increase in K+ concentration in medium and reduce the level of activity. This mini sensor is also able to discriminate between Li+, Rb+ and K+ ions like the wild-type KdpD. A plasmid coding for this mini sensor allows a kdpD deletion strain to grow under K+-limited conditions in medium. Presumably, the signal perceived by KdpD is in the periplasm, but how is the signal transmitted to the cytoplasmic domain of KdpD that controls activity? Perhaps KdpD is not the direct sensor, an additional component in the membrane might sense the signal and communicate with KdpD. Further research with sodium carbonate extraction to determine the membrane localisation of C499-894 showed the protein mainly found in the supernatant, suggesting C499-894 is a soluble protein, although C499-894 could be attached to the membrane to come in contact with an unknown membrane protein. Looking for the unknown component two protein candidates were found by co-purification, the cytochrome oxidase A (CyoA) and the glucosamine-6-phosphate synthase (GlmS). To find an interaction between these proteins and KdpD more research has to be done.
Olfaktorische Rezeptoren mit speziellen topographischen Expressionsmustern
(2007) Feistel, Torben; Breer, Heinz
In the present study, olfactory receptors (ORs) featuring special expression patterns in chemosensory subsystems were analyzed. The data showed that out of a repertoire of about 1000 ORs a restricted group (about 50) was not only expressed in the olfactory epithelium but also in the vomeronasal organ, which is generally defined by the expression of characteristic V1R and V2R receptors. The ?ectopically? expressed OR genes represent different receptor families including genes from gene-clusters located on different chromosomes. However, in all individuals the same set of genes seemed to be expressed. The majority of the expressed ORs was present in only a small number of VNO cells, however some were found to be expressed in more than 100 cells. One distinct OR gene (mOR261-6) was expressed in many more cells of female VNOs than in males. The highest number of OR expressing cells was observed in a short postnatal, pre-pubertal period. Cells with mOR18-2-receptors were also found to express a V1R gene. In these cells the OR18-2 gene did not show a mono-allelic expression as compared to expression in the main olfactory system but rather was transcribed from both alleles (bi-allelic) in the vomeronasal neurons. The functional implication of receptor coexpression and bi-allelic OR-expression are unknown. Receptors of the OR37 gene family are exclusively expressed in the olfactory epithelium, in which they are expressed in a special pattern in a central area of the nasal turbinate. Detailed analysis of the spatial distribution of cells equipped with distinct OR37 subtypes revealed that these cells were positioned in specific sub-compartments within this central region. The high number of OR37 expressing cells resulted in the lower number of cells with receptors which are zonally distributed. This implies that cells in a small circumscribed area preferentially expressed OR37 genes. The mechanisms underlying this unique spatial expression pattern of OR37 genes in the olfactory epithelium are currently unknown. A newly generated mouse line was arranged in which even very transient transcription of a OR37 gene is visualized by permanent label in the expressing cells. The examination showed that cells outside of the OR37 cluster did express a OR 37 family member, yet transcription in these cells was rapidly terminated. These results suggest a mechanism which allows an initial transcription of these genes in different areas of the epithelium, but a sustained expression of OR37 genes is restricted exclusively to the central recess of the turbinate. The special features of the OR37 subtypes have led to the hypothesis that these receptors possibly react to chemical compounds relevant for mammalian species. Thus complex mixtures of volatile compounds from the habitat of mice were examined. Electroolfactograms from different regions of the olfactory epithelium showed that the ?head-space? of the embedding from mice cages elicited a stronger response in the central area of the turbinate IIb than in areas outside of this region. Single substances out of this complex mixture induced similar responses in different epithelial recesses. However, 6-hydro-6-methyl-3-heptanon elicited stronger responses within the central OR37 expressing region. This substance is considered to be a mouse pheromone. Taken together, these data on expression and ligand specificity of distinct olfactory receptors suggest, that the vomeronasal organ and the olfactory epithelium although structurally separated chemosensory systems, overlap in their response, spectrum and in their functions, particularly in detecting compounds with biological relevance.
Vergleichende Untersuchungen zur Musterbildung in erregbaren Medien mit Vermerken zum Einfluss schwacher magnetischer Felder - Schwerpunkt: Belousov-Zhabotinsky-Reaktion
(2007) Dolzmann, Kerstin; Hanke, Wolfgang
In this work we did some research on the influence of a weak external magnetic field (MF) on the creation of patterns in excitable media (duration field (DC) and alternating field (AC)). As examples we chose the well known Belousov-Zhabotinsky reaction (BZR) and ferrofluids. If ferrofluids are stimulated mechanically by vertical vibration they show changes of phases in the building of patterns while raising the induced energy (here by different hights of amplitudes). The viscosity of the magnetic fluid is increasing in a DC-field. Because of this the changing of the phase is different from the ones without an external force. A non-stationary stirred BZR shows a periodic change of colour between yellow and colourless ? or red and blue, if ferroin is added as a catalyst. This oscillation is described as a simple curve in literature. We, however, found a much more complex behaviour in the experiment. The intrinsic optical signals (IOS) of a ferroin-catalysed, stirred BZR show a double-peak at the beginning of the reaction, which is fading after a few further oscillations. This behaviour depends on the concentration of ferroin and resembles very much electrical and optical signals known from neuronal processes (e.g. retinal spreading depression). This basic similarity makes the BZR an ideal model for a variety of neurophysiological signals, even if the underlying mechanisms are completely different. If this system is put to the influence of a weak external magnetic DC-field a further inner oscillation is added to the double-peak behaviour of the IOS. Also the double-peak itself looks different from the one without external field. If further components are added to the system it gradually changes to chaotic behaviour. This could be the induced little currents in an AC-field. Each expansion of the system is followed by further bifurcation culminating in a transition from pattern to chaos. BRZ gels show bright propagating concentric rings or spirals as a pattern. With the used materials and methods of measurement we were not able to record changes in the formation of patterns if the system was expanded. But one can assume that the behaviour of the gel is changing in the DC-field: all in all it seems to get faster.
Lokalisation von Pheromon-Rezeptoren und -Bindeproteinen in antennalen Sensillen von Insekten
(2007) Gohl, Thomas; Breer, Heinz
The remarkable reactivity of moth to specific pheromones is based on the extreme selectivity and sensitivity of sensory cells in the male antennae. This feature is supposed to be based on cells equipped with specific receptors. Only the sequencing of the genomes of Bombyx mori and Heliothis virescens provided the possibility to identify candidate genes of olfactory receptors in moths. Upon detailed inspection of candidate receptors it turned out that within the generally very heterogeneous group of receptor-genes a subfamily exists containing members of both species showing striking sequence homology. A conservation of the primary structure of receptors for pheromones has been postulated. For a continuing characterization in these studies different approaches were used to verify that these receptors are indeed expressed in cells of pheromone-sensitive sensilla (sensilla trichodea). By means of ?whole mount? in situ hybridization experiments the RNA of the receptor types BmOR1 and BmOR3 could be visualized in directly neighboring cells reflecting the topology of trichoid sensilla. Also some of the Heliothis receptor types (HR13, HR14, HR16) could be assigned to sensilla trichodea. In addition to the specific receptors, the pheromone binding proteins (PBPs) are expected to play an important role in the detection of hydrophobic pheromone molecules. PBPs are produced by glia-like cells surrounding the sensory neurons. In double in situ hybridization experiments it could be shown that HR13-cells are indeed surrounded by cells expressing HvirPBP1 and HvirPBP2. Analysis comparing the topology of different receptor-types showed that cells expressing HR13 can be assigned to sensilla trichodea type A, whereas HR14 and HR16 are expressed in cells of sensilla trichodea type C. This characteristic expression pattern is considered as a further indication that these candidate-receptors are indeed pheromone-receptors. The assignment of individual receptor-types to distinct sensilla-types provides the basis for investigating the functional implications of receptor-types for the registration of main or minor components of complex pheromone-blends. Further it turned out that HR13 shows coexpression with SNMP1 (sensory neuron membrane protein 1) which is considered as a ?marker?-protein for antennal sensory neurons. This is however not the case for receptor types HR14 and HR16. In search of further SNMP-types screening-experiments were carried out which led to the identification of a novel SNMP-type (SNMP2) of Heliothis virescens. Subsequent studies concerning the expression of SNMP2 showed that the topologic distribution of SNMP2-cells is comparable to SNMP1-cells, but they show a different morphology. Further experiments revealed that SNMP2 is in fact expressed in PBP-producing cells. These findings imply that the proposed putative function of SNMPs has to be reconsidered. One major goal of this study was the attempt, to identify receptor-relevant cells by visualization of mRNA via in situ hybridization but to visualize the localization of the receptor-protein via immunohistochemical approaches. Although the generation of antibodies for olfactory receptors is very difficult, it was possible to raise antibodies specific for receptor type HR13. Using these antibodies in immunohistochemical approaches allowed to also visualize HR13-receptor-protein. By means of double-staining experiments using HR13-specific antisense RNA-probes and anti-HR13 antibodies mRNA and protein were visualized in the same specific cells. Using confocal laserscanning microscopy, it was possible to document that receptor-protein was indeed located in the sensory dendrites. Further, the receptor-protein was also visualized in the axonal processes of sensory cells and the receptor-specific staining revealed that within the antennal nerve HR13-axons appear to be organized in fascicles. These HR13-immunolabeled fascicles were visible until they reach the ?sorting zone? of the antennal lobe; in contrast to mouse olfactory bulb, no receptor specific staining was visible in the antennal lobe.
Analyse relevanter Signalwege der strahleninduzierten COX-2 Expression in Tumorzellen
(2007) Krebiehl, Guido Klaus; Rodemann, H.-Peter
Summary: Cancer is a health problem worldwide and the number of new cases is rising. Surgery, radiotherapy and chemotherapy are the major treatment modalities. New developments in radiotherapy make radiation alone and in combination with chemotherapy to an important therapy becoming more and more mattering. The success of a therapy often depends on the genetic profile of a tumor. This makes analysis of molecular processes in cells after radiation an important aspect in radiotherapy developing an effective strategy for tumor treatment. COX-2 is overexpressed in a lot of tumors and correlates with a poor prognosis. Moreover COX-2 can be induced by ionizing radiation. This makes COX-2 an interesting molecular target in radiation therapy and in cancer therapy in general. Studies with specific COX-2 inhibitors came to different results in different cell lines. The aim of the presented study was to investigate the survival and the proliferation of prostate cancer cells after treatment with ionizing radiation alone and in combination with specific COX-2 inhibitor Celecoxib and the analysis of signaling pathways leading to radiation induced COX-2 expression. The following major results were obtained: 1. Treatment with Celecoxib had no influence on the radiosensitivity of the prostate cancer cell lines investigated. 2. The proliferation of different cell lines was inhibited by the treatment with Celecoxib. 3. The inhibition of the proliferation seems to be independent of the level of COX-2 of the cell lines. 4. Apoptosis can not be induced by Celecoxib in clinical relevant doses in the cell lines investigated. 5. Induction of COX-2 expression by ionizing radiation depends on the cell line investigated. 6. The MAPK-signaling pathways play a major role at COX-2 expression. In conclusion the results of the presented study indicate that COX-2 can be an important molecular target in radiation therapy. Although this depends on the cell line investigated. As well, the signaling pathways leading to a radiation induced expression of COX-2 are individual for each cell line. Thus the application of Celecoxib during radiation therapy can be positive on the treatment of different tumors.
Einfluss von erhöhtem atmosphärischen CO2 auf die N2-fixierende Symbiose von Trifolium repens L. und Rhizobium leguminosarum biovar trifolii
(2007) Stöber, Sara; von Wirén, Nicolaus
CO2 is one of the main greenhouse gases strongly influencing the climate and the terrestrial ecosystem. Up to know little is known about the impact of elevated atmospheric CO2 on symbiotic interactions in the rhizosphere, especially on the N2-fixing symbiosis between Trifolium repens and Rhizobium leguminosarum biovar trifolii. First results of a ten-year Free-Air CO2 enrichment experiment (Swiss FACE) showed that after three years of CO2 fumigation the genetic composition of the Rhizobium population in the root nodules of T. repens had changed. The first part of this thesis set out to clarify the question whether a genetic difference in the Rhizobium population of root nodules of white clover could still be detected after ten years of CO2 fumigation or if an adaptation of the nodule bacteria to elevated CO2 concentrations had occurred. Furthermore the thesis addressed the question whether elevated atmospheric CO2 leads to quantitative and qualitative changes in the root exudation of T. repens particularly with regard to exudation of signal substances during the nodulation process. In summer 2002 white clover plants were collected from plots fumigated with CO2 and control plots of the Swiss FACE. Rhizobium strains were isolated from the clover root nodules and used for rep-PCR DNA fingerprinting. Results clearly showed that after ten years of CO2 enrichment changes in the genetic composition of the R. l. bv. trifolii could no longer be observed. Thus, CO2-induced changes in the population structure of rhizobia seemed to be transient. This can be traced back to the possibility that over the experimental period a new C/N equilibrium in the grassland ecosystem has been established. At the beginning of the FACE experiment an increase in the C/N ratio of the soil was detected, which could be balanced in the course of time through enhanced symbiotic N2 fixation and consequently a higher N input into the ecosystem. The observed stabilisation of the grassland ecosystem most likely caused a reduction of the indirect CO2 impact on the microorganisms. This might explain why a change in the genetic composition of Rhizobium strains was not longer detected after ten years in the Swiss FACE. To investigate an influence of elevated atmospheric CO2 concentration on the release of signalling compounds clover plants were cultivated hydroponically in two independent climate chamber trials under axenic and non-axenic conditions at ambient and elevated CO2 concentrations (400 and 800 ppm) and different levels of N supply. Root exudates were collected over a period of seven hours and at three and four different plant ages, respectively. Phenolic compounds were extracted by solid-phase extraction and afterwards analysed with HPLC and LC-MS. Additionally, the isolated fractions were tested for their ability to induce the nodulation genes of R. l. bv. trifolii using a nod-gene induction test. The CO2 enrichment caused an increase in shoot and root growth in both experimental setups, but did not provoke a change in the C/N ratio of the roots. Besides the known signal compound 7,4?-dihydroxyflavone new phenolic substances could be detected, which have not yet been described in literature. The fractions were identified by their polarity, light absorption and molecular weight as aglyca and flavones. All of these had the ability for nod-gene induction except one fraction (fraction 2). CO2 influenced the exudation of signalling compounds quantitatively but not qualitatively. The enhanced exudation, especially of 7,4?-dihydroxyflavone, could be attributed to the higher root mass under elevated CO2 but also to a higher release rate on a root fresh weight basis. The CO2 reaction of the clover plants, for the biomass production as well as for the root exudation, was clearly dependent on the N supply and only significant under axenic conditions. In individual cases the N impact was more pronounced than the CO2 effect: with increasing N demand axenic clover plants enhanced the exudation of the nod-gene inducing fraction C. It is concluded that this fraction, identified as a hydroxyflavone, has therefore an important signal function under N limitation. Besides the CO2 concentration and N supply, root exudation by T. repens was considerably influenced by the plant age, which caused a reduction of the signal exudation in older plants and qualitative changes of the released phenols, especially under non-axenic conditions. The present study suggests that the genetic shift of R. l. bv. trifolii detected at the beginning of the Swiss FACE experiment was most likely a consequence of the enhanced exudation of phenolic signal compounds of T. repens under elevated atmospheric CO2 concentrations.
Regulatory elements controlling the expression of OR37 genes
(2007) Zhang, Yongquan; Breer, Heinz
The genes of the OR37 family are clustered in two loci (cluster I and cluster II) on mouse chromosome 4. These genes encode distinct olfactory receptors (ORs) which are characterised by an insertion of six amino acids in the third extracellular loop and moreover, these receptor types are only expressed in cells which are segregated in a small patch on the central nasal turbinate. As first steps to unravel the molecular basis of this unique topographic expression pattern previous studies have led to the identification of highly conserved sequence motifs including an olf-1 site in the putative promoter region of these genes and subsequently several transcription factors were identified which did bind to these sites. However, it remained elusive if an interaction between the transcription factors and the putative promoter sites may have functional implications. Therefore, a heterologous system was employed to assess the consequence of an interaction between the putative promoters and the transcription factors. HEK 293 cells were cotransfected with a reporter gene under the control of putative mOR37 promoter regions and an expression vector based gene encoding the transcription factor. The expression rate of the reporter gene was monitored by measuring luciferase activity. It was found that the three O/E transcription factors (O/E-1, O/E-2 and O/E-4) induced significant activation of the mOR37 promoters; in addition, it was observed that the putative promoters of other OR genes were also activated, suggesting that the O/E proteins may play a general role in the regulation of OR gene expression. Mutagenesis experiments revealed that the effects of O/E proteins were dependent on the presence of an olf-1 site within the promoter region. For the transcription factor Lhx-2 it was found that not all but only promoters of distinct OR-genes were affected. For the mOR37 promoters a simultaneous action of O/E protein and Lhx-2 elicited an increase of reporter gene expression. The data indicate that the putative mOR37 promoters could drive gene expression in the presence of the crucial transcription factors in this heterologous system. In order to explore to what extent the promoter may contribute to the characteristic topographic expression pattern of the mOR37 genes in vivo, a mOR37C transgene which included the coding exon and the putative promoter, was randomly inserted into the mouse genome. Seven lines were obtained; in all lines the transgene was specifically expressed in olfactory sensory neurons (OSN). In six lines the transgene expression was restricted to the central patch of the olfactory turbinates, typical for the OR37 genes. In one line (line 7) the transgene was also expressed in OSNs ectopically positioned outside the patch within the medial zone. It was found that the transgene was expressed in a mutually exclusive manner and from only one allele. The axons of OSNs expressing the transgene co-converged in the same glomerulus with the axons from neurons expressing the endogenous gene. In line #7 the formation of ectopic glomeruli was observed. The number of OSNs expressing the transgene varied considerably among lines; these differences were independent from the copy number of the transgene. The data indicate that the short putative promoters, most likely the conserved motifs, were sufficient to drive the OR37 gene expression in a tissue specific way and most aspects of the OR37 gene expression were mimicked by the transgene; however, considerable differences between certain lines suggested additional regulatory elements, such as a locus control region (LCR). Since regulatory elements for gene transcription, such as promoters, enhancers and LCRs, appear to be conserved across species, a comparative approach was utilized to search for the LCR-like element for the OR37 locus by sequence alignment across distantly related mammals. A segment of 270 base pairs located 137 Kb upstream of OR37 cluster I was found to be highly conserved between mouse, human, dog and opossum. It was not associated with an exon of any known gene and was highly correlated with OR37 cluster I rather than with the neighboring genes, since the flanking genes did not show syntenic conservation in the opossum genome. A homologous counterpart for this segment was found downstream of the OR37 cluster II locus; an alignment of the cluster II sequence across species identified the conservation of this counterpart. Examination for relevant motifs in this segment and comparison with the conserved H element revealed two common transcription factor binding sites, at least one of them is known to be essential for generating DNase I hypersensitive sites in the LCR of the beta globin gene locus. Further studies are required to evaluate a possible role of this conserved segment in the regulation of the OR37 gene expression.
Carbon and nitrogen transformations in alpine ecosystems of the Eastern Alps, Austria
(2007) Koch, Oliver; Kandeler, Ellen
This thesis investigated net CH4 and net CO2 emissions from sites in the alpine region of the Eastern Alps, Austria. Four mature alpine sites (one dry meadow and three fen sites) were selected and the influence of abiotic (radiation, temperature, soil water conditions) and biotic (above-ground standing plant biomass) environmental controls on diurnal and seasonal emission patterns were studied. For a better understanding of the response of soil C- and N pools to global warming, the temperature sensitivity of activities involved in C- and N cycling were determined. The first part of the thesis dealt with net methane fluxes measured over a period of 24 months. During snow-free periods, average methane emissions of the fen sites ranged between 19 and 116 mg CH4 m-2 d-1. Mean emissions during snow periods were much lower, being 18 to 59% of annual fluxes. The alpine dry meadow functioned as a small methane sink during snow-free periods (-2.1 mg CH4 m-2 d-1 (2003); -1.0 mg CH4 m-2 d-1 (2004)). The diurnal and seasonal methane uptake of the dry meadow was positively related to soil temperature and negatively related to water-filled pore space (wfps). In the fen, the seasonal methane fluxes were related to soil temperature and groundwater table. The live above-ground standing plant biomass contributed to net methane fluxes only at those sites with higher water table positions. This study provided evidence that alpine fens acted as methane sources throughout the year, whereas an alpine meadow site acted as a net methane sink during snow-free periods. In the second part of the thesis the CO2 balance was estimated based on diurnal flux measurements and on the influence of photosynthetic active radiation (PAR), plant green area index (GAI), soil temperature and wfps. The daylight net ecosystem CO2 emission rate was influenced by PAR and GAI throughout snow-free seasons. The seasonal net CO2 emission rate at night was positively related to soil temperature, while low wfps reduced flux rates at the meadow and at the driest fen study site but reinforced carbon loss at the wetter fen sites. The daily average ecosystem net CO2 gain during snow-free periods at the meadow was 3.5 g CO2 m-2 d-1 and at the fen sites between 1.5 and 3.4 g CO2 m-2 d-1. The mean average daily CO2 emission during snow periods was low, being -0.9 g CO2 m-2 d-1 for the meadow and between -0.2 and -0.7 g CO2 m-2 d-1 for all fen sites. All sites function as significant annual net carbon sinks, with a net carbon gain from 50 to 121 g C m-2 a-1 (averaged over both years), irrespective of water balance. The results indicate that alpine fen sites, that have built up a large carbon stock in the past, are not expected to gain a further carbon surplus compared with meadows under the current climate. Temperature is important for regulating biological activities. The third part of the thesis focused on temperature sensitivity of soil C mineralization, N mineralization and potential enzyme activities involved in the C- and N cycle (ß-glucosidase, ß-xylosidase, N-acetyl-ß-glucosaminidase, tyrosine aminopeptidase, leucine aminopeptidase) over a temperature range of 0-30°C. The objective was to calculate Q10 values and relative temperature sensitivities (RTS) and to quantify seasonal (summer, autumn, winter) and site-specific factors. The Q10 values of C mineralization were significantly higher (average 2.0) than for N mineralization (average 1.7). The Q10 values of both activities were significantly negatively related to soil organic matter quality. In contrast, the chemical soil properties, microbial biomass and sampling date did not influence Q10 values. Analysis of RTS showed that the temperature sensitivity increased with decreasing temperature. The C- and N mineralization and potential aminopeptidase activities (tyrosine, leucine) showed an almost constant temperature dependence over 0-30°C. In contrast, ß-glucosidase, ß-xylosidase and N-acetyl-ß-glucosaminidase showed a distinctive increase in temperature sensitivity with decreasing temperature. Low temperature at the winter sampling date caused a greater increase in the RTS of all activities than in autumn and summer. Our results indicate a disproportion of the RTS for potential enzyme activities of the C- and N cycle and a disproportion of the RTS for easily degradable C compounds (ß-glucose, ß-xylose) compared with the C mineralization of soil organic matter. Thus, temperature may play an important role in regulating the decay of different soil organic matter fractions.
Zelluläre Mechanismen beim Neuro Tissue-Engineering
(2007) Dreesmann, Lars; Schloßhauer, Burkhard
To date, numerous tissue engineering approaches aim to develop artificial nerve guide implants for the treatment of lesioned nerves. However, the gold standard in this therapeutical application is still the autologous nerve transplantation. By allocation of a native structure, comprising Schwann cells as well as endothelial cells, an optimal supply of regenerating axons is warranted. Because this surgical procedure retrieves several risks, intensive research is done worldwide to develop alternatives. In this context, Schwann cells play an important role. By migration along the lesion site, they built up guidance rails, so called bands of Büngner. These bands offer an ideal substratum for regrowing axons. However, antagonistic fibroblasts may hamper these events. Hence, the interaction between the two cell types was analyzed in detail, setting a focus on migration. Results indicated that fibroblasts foster Schwann cell migration. Neuregulin was identified as molecular mediator causing this effect. Further experiments revealed that neuregulin promotes Schwann cell migration via erB-receptor and the RhoA pathway. With respect to the concept of a nerve guide implant this means, that the inner membrane should allow diffusion of growth factors, but exclude regeneration inhibiting fibroblasts from the inside. For this purpose, gelatin membranes were characterized regarding their physical and chemical properties. Cell biological testing with gelatin tubes shed light on permissivness for Schwann cells, semipermeability for nutrients and exclusion of fibroblasts. Because a better supply with nutrients promises an additional acceleration of regeneration, the formation of blood vessels next to the implant should be promoted by a gelatin sponge. The immigration of blood vessel forming endothelial cells was analyzed using immunocytochemistry and microscopy. Neovascularisation, biocompatibility and inflammation were investigated on the chorioallantoic membrane of the chicken egg, as well as with subcutaneous implantation into mice. Implantation of gelatin nerve guide tubes in lesioned rat sciatic nerves showed an increased angiogenesis and hardly any inflammation. Within the scope of this work, the application of several molecular and cell biological assays, in combination with three different animal models, made a contribution to the detailed comprehension of mechanisms during nerve regeneration, together with an interdisciplinary bridging between material science and biology in development of innovative therapeutical approaches.
Neuronale Modulation : der Einfluss von Agonisten und inverser Agonisten auf das Cannabinoidsystem einer hippocampalen Primärkultur
(2007) Klink, Oliver; Hanke, Wolfgang
The aim of this thesis was to investigate the effect of inverse agonists on the CB1-receptor with an established complex neuronal-/ glia- co culture obtained from hippocampi of embryonic rats. To provide evidence of the expression of the CB1-receptors in the established culture immunocytochemical studies have been used and showed a sufficient expression level of the CB1-receptors. The neuronal culture was further tested on various electrophysiological parameters to verify an in vitro assay that resembles in vivo characteristics. Thus the exposure of TTX to neurons lead to reduced spike activity which refers to the blocking of voltage gated sodium channels. Also the inhibition of AMPA receptors using CNQX showed a reduction of spike activity in respect of the reduced synaptic activity. Analyses of the kinetic and spike frequencies of the generated actionpotentials as well as the kinetics and frequencies of spontaneous AMPA- and NMDA epscs are to a large extent comparable to published data of in vitro and in vivo assays. To reduce the intrinsic variability of the established cannabinoid assay the method of induced burst activity under low magnesium conditions has been used. This method results indeed in a lower variability of the assay but also the analysis of the effect of cannabinoid agonists and inverse agonists on the evoked burst activity showed interesting inverse modulations of burst durations, event intervals and inter-event intervals compared to alterations of the spike frequencies. On the basis of the obtained data by the analyzed spike-frequencies an agonistic effect of nanomolar concentrations of the investigated inverse agonists could be shown for the first time. This observation could lead to the conclusion that this might be a specific interaction of the investigated inverse agonist with an other receptor. The inhibition of the adenylatcyclase, a key-enzyme of the CB1 signal transduction, neutralizes the agonistic effect, although the inverse agonistic effect dissapeard. In addition the interaction of the opioid system in respect to the observed agonistic effect of rimonabant has been investigated. However, non of the published interaction of this two systems in respect to the agonistic effect of rimonabant could be observed.
Interaktion des Photosensors Ppr aus Rhodocista centenaria mit Proteinkomponenten der chemotaktischen Signaltransduktion
(2008) Kreutel, Sven; Kuhn, Andreas
Rhodocista centenaria (previously known as Rhodospirillum centenum) is a photosynthetic alpha-proteobacterium which exhibits a unique phototactic response in respect of the direction of light. In this work, the focus is on the potential photoreceptor Ppr and its C-terminal histidine kinase Pph to identify putative binding partners in the signal transduction pathway. The results of overexpression experiments with the Ppr-receptor or the Pph-domain in E. coli indicated that there may be an interaction between the photosensor and the chemotactic signalling pathway. Even cells expressing only small amounts of the R. centenaria proteins showed no chemotactic response at all, whereas uninduced cells exhibited normal chemotactic response on swarm plates as well as in capillary assays. To investigate whether the receptor interacts with components of the chemotactic pathway, the Ppr-protein and the Pph-domain as well as the chemotactic proteins CheW and CheAY of R. centenaria were heterologously expressed in E. coli and purified to homogeneity by affinity chromatography. Binding experiments were carried out by using an IAsys biosensor cuvette system. The results indicated that the kinase domain Pph binds to the chemotactic linker protein CheW with a dissociation constant of 0.13 ± 0.026 μM. Pull-down experiments were made to verify this finding and to investigate the role of ATP in the binding process. The results confirmed our previous observations but in contrast to the complex formation in the E. coli chemotactic pathway, the binding of the C-terminal histidine kinase to CheW was ATP-dependent. To study whether the kinase domain also binds CheW in vivo, expression and coelution experiments with tagged Pph-protein were carried out in R. centenaria. The findings suggest that the complex formation occurs in vivo as well as in vitro. From the data of the E. coli chemotactic complex formation it is well known that CheA is part of the trimeric complex which consists of MCP-receptors, CheW and CheA. To analyse this, pulldown experiments with all three proteins (Pph, CheW and CheAY) were performed. The results clearly showed a participation of CheAY in the formation of the complex with CheW and the kinase Pph. It is known that the photoreceptor Ppr is autophosphorylated during its light induced photocycle. We therefore examined whether the kinase domain is sufficient for this autophosphorylation reaction and whether CheAY could function as a phosphate acceptor. Our results confirmed the hypothesis that the kinase domain is sufficient for autophosphorylation and that the Pph-protein assists CheAY to take over phosphate groups. Taken together, the results in combination with data from the literature lead to a detailed working model for the function of the photoreceptor Ppr and the signal transduction pathway.
Untersuchungen zur räumlichen Heterogenität von Kronenstruktur und Bestandesniederschlag in einem tropischen Bergregenwald
(2008) Oesker, Mathias; Küppers, Manfred
The objective of this study was to investigate the distribution and heterogeneity of canopy structure and precipitation throughfall and related with this the factors light, water, and nutrient input. Further to appraise the consequences of this heterogeneity as a factor, which forms ecological niches. The study took place in a tropical mountain rain forest in southern Ecuador, specifically in the Reserva San Francisco located north of the Podocarpus National Park. The area of research covered the altitudinal range from 1950 m a.s.l. to 2275 m a.s.l. Nine plots of 400 m2 (20 m by 20 m) were set up in three forest types, which differed in tree species composition (HOMEIER 2004). Two forest types were located on a ridge and one was in a gorge at the same elevation as the lower ridge forest type. In each forest type three representative plots were chosen and a total of 31 study points were defined. At each point throughfall was collected during a one-year period. In all throughfall samples the following parameters were determined: Volume, pH, electrical conductivity, concentration of K, Mg and Ca, nitrate, ammonium, organic nitrogen, phosphate, P, Mn, Cu, Rb, Sr and Pb. The canopy structure was determined at all points with both structural measurements and hemispherical photography over a period of three years. Lab experiments with a representative selection of tree species were performed in order to determine leaf surface water storage capacity and nutrient leaching out of leaves. For determination of canopy structure hemispherical photography turned out to be a particularly efficient method. The software HemiView (DeltaT) was used to calculate important information such as canopy openness, light environment and LAI. A high spatial heterogeneity with a coefficient of variation (CV) of 59 % was found for all parameters. It was higher than the temporal variability over three years (CV 12 %). The throughfall was most heterogeneous within the investigated parameter with a CV of 64 %. In total close to 82 % (between 0.5 % and 492 % and a CV 29 %) of the volume of the incident precipitation could be collected as throughfall in the forest. With this throughfall 49.1 kg ha-1 a-1 K, 3.7 kg ha-1 a-1 Mg and 8.7 kg ha-1 a-1 Ca (mean values) were transported. During low intensity rain events the proportion of throughfall, expressed as percentage of incident throughfall was significantly lower than the annual mean of the incident precipitation. For high rain intensities no differences were found. With a geostatistical approach to investigate the spatial distribution of the throughfall no clear results could be calculated because the three replicates diverged strongly from each other. Canopy structure and its species composition was influenced by the distribution of throughfall. Related to the amount of throughfall it could be shown that with an open canopy up to 100 % of the incident precipitation could be collected. Underneath a closed canopy, in average less throughfall was collected. However, the volume of throughfall showed high spatial distribution and heterogeneity with even more than 100 % of incident precipitation. Nevertheless, throughfall volume can be predicted using the parameters radiation and canopy openness at a zenith angle of 36°. Average water storage capacity of leaf surfaces from eleven most common tree species resulted in 74.74 ml m-2 leaf area. In a dry canopy with a theoretical equal distribution of precipitation and a given LAI this value equals 0.38 mm of rain. The nutrient leaching out of leaves is species dependent and differs statistically. Those lab results can be extrapolated to the entire forest: Including the water storage capacity and the number of rain events, a maximum leaching capacity of 220 kg ha-1 a-1 K, 14 kg ha-1 a-1 Mg and 67 kg ha-1 a-1 Ca can be calculated. The main focus of this study was to investigate heterogeneity of abiotic factors and its ecological consequences. In the forest type with the most heterogeneous canopy structure and the most heterogeneous distribution of throughfall amounts were found. Lowest heterogeneity of spatial distribution of throughfall element contents was found in forest type with the lowest tree species diversity. The higher the tree species diversity the more heterogeneously scattered is the element content in the throughfall.
Deskriptive und funktionelle Analyse der Mitglieder der Calponin-Genfamilie Xclp1, Xclp2 und Xclp3 während der Embryonalentwicklung von Xenopus laevis
(2008) Schmalholz, Silke; Blum, Martin
The embryonic development of vertebrates is characterized by controlled cell movements. During gastrulation and neurulation cells of the presumptive heart tissue and the neural crest after neural tube closure migrate towards their final position in the embryo. Cell intercalations, which drive the convergent extension (CE) movements to elongate the embryo also depend on active cell migration. The inhibition of CE leads to shortened body axis and neural tube closure defects (NTD). The motility of eukaryotic cells is finally based on the dynamic interaction of cytoskeletal components, which act on the actin filament. Secreted growthfactors of the Wnt family can regulate embryonic cell movement via the non canonical Wnt signaling pathways. The planar cell polarity (PCP) and the Wnt/Ca2+ pathway are thought to be crucial for the process of CE. Up to now there is a lack of knowledge about the cytoskeletal effectors of these signaling cascades. In the presented work, members of the calponin gene family (clp1 to 3) were analysed in this context. Calponins are actin binding proteins, which have been shown to inhibit actin-myosin-interactions and/or to stabilize the actin filament. Expression patterns provided first insights in the transcriptional activity of Xclp1, Xclp2 and Xclp3 during embryonic development. Two Xclp genes (Xclp2 and Xclp3) were already expressed broadly at the onset of gastrulation. Transcription, however, was not detected in the involuted cells, which form the mesodermal germlayer. At neurula stages Xclp2 mRNA was specifically found in the notochord, whereas Xclp3 was expressed in the neuroectoderm. Additionally the migrating cells of the embryonic heart and neural crest were positive for calponin expression. In summary the embryonic calponin pattern correlated with tissues in which cell movements occur. Over- or misexpression experiments were performed to manipulate embryonic calponin function in Xenopus laevis. Gain of function experiments however did not interfere with embryonic development. Probably calponin function was posttranslational negatively regulated in these experiments.The overexpression of calponin proteins, in which specific phosphorylation sites were mutated or known regulatory calponin domains deleted, again didn´t result in altered phenotypes. However, the misexpression of calponin actin binding domaine 2 (ABD2) inhibited the migration of Krox 20 positive neural crest cells, suggesting that in this tissue the Xclp ABD2 acts dominant negative. The presented data are not able to proof or disproof the hypothesis, that calponin proteins are effectors of the non canonical Wnt pathways.
Analyse von Pathogenresistenzmechanismen in Tomate (Solanum lycopersicum L.)
(2008) Gerhardts, Anja; Pfitzner, Artur J. P.
For many organisms plants serve as a source of nutrients and energy, but because of their static location they are exposed to various harmful environmental influences. Due to this factor they have developed complex defence mechanisms e. g. for protection against pathogens. An important aspect of these defence mechanisms is the expression of intrinsic resistance genes (R) that detect pathogenic avirulence gene products (Avr) thereby causing a hypersensitive response (HR) in the infected cells and consequently inhibiting the systemic infection of the plant. In this work the resistance genes Tm-2 and Tm-2² of tomato were isolated, cloned and sequenced. The allelic R genes are members of the CC-NBS-LRR group of resistance genes, which is widely spread in plants, and differ only in four amino acids. This is surprising because using resistance breaking ToMV strains Weber et al. (2004) showed that both resistance gene products interact differently with the movement protein (30 kDa MP = Avr) of the virus. To gain further insight into this phenomenon of different pathogen detection, chimeric exchange constructs (A1 and A2) were designed through restriction in the region between the NBS and the LRR domain. These four constructs were used for transformation of MM tomatoes as well as NN and nn tobacco plants. The expression of the resistance gene constructs in MM an nn lines did not confer the expected resistance to ToMV. Nevertheless in older infected nn transformants a formation of spontaneous necrosis was observed, which indicates a delayed development of HR. One possible explanation could be that the presence of only the resistance gene product is not sufficient to detect the viral movement protein and that other host cellular components are involved in this process (as in the guard hypothesis by Dangl and Jones, 2001). This assumption is supported by our yeast two hybrid interaction experiments which showed that a direct interaction of Tm-2 and 30 kDa MP can be excluded. For the NN transformants differences in functionality of the constructs was observed. While NN/Tm-2 and NN/A2 plants showed extreme resistance to ToMV wild type (ToMV0) and the Tm-2² resistance breaking strain ToMV2², the Tm-2² and A1 constructs conferred less resistance to ToMV0 and the Tm-2 resistance breaking strain ToMV1-2. This finding also supports the assumption that there is a difference in pathogen detection between the two alleles. Furthermore it shows that the detection takes place within the LRR region because the exchange construct that behaves in the same way as the endogenous resistance gene carries the C-terminal LRR domain of this allele. The hydroxycinnamoyl-CoA:tyramine N-(Hydroxycinnamoyl)transferase (THT) was found to be another candidate for transmission of pathogen resistance during HR (Gerhardts, 2003). Our in vivo results show that the products of the THT enzymatic reaction induced during HR does not only have an antimicrobiotic effect on the pathogen (von Roepenack-Lahaye et al., 2003; Newman et al., 2001) but also has an apoptotic effect on the plant cell itself.
Identifizierung und Charakterisierung der Succinsemialdehyd-Dehydrogenase aus parasitischen und nichtparasitischen Arthropoden
(2008) Rothacker, Boris; Ilg, Thomas
The objective of the PhD project was the molecular and enzymological characterization of SSADH in parasitic and nonparasitic insects and acarids, organism groups where this enzyme was virtually unexplored. In this project, the list of investigated non-arthropod organisms included the bacterium Escherichia coli (Ec) and the mammal Mus musculus (mouse). Their SSADH served as reference enzymes or were used for biophysical experiments. Amongst the arthropods, Drosophila melanogaster (Dm) (fruit fly) was chosen as nonparasitic model insect. Lucilia cuprina (Lc) (sheep blowfly) and Ctenocephalides felis (Cf) (cat flea) were included as important parasitic insects, as was the acarid Rhipicephalus microplus (Rm) as the economically most important tick species. At the start of this thesis, expressed sequence tag studies and whole genome sequencing on Dm suggested the existence of a single copy gene candidate that, based on amino acid sequence homology, was considered to be a candidate for a SSADH ortholog in this species (DmSSADH). The Dm SSADH gene candidate was cloned and expressed in Ec as a soluble protein. To compare the enzymological properties of DmSSADH, another so far uncharacterized Dm gene candidate for an acetaldehyde dehydrogenase (DmALDH) was cloned and expressed in Ec as a soluble protein. Both expression products showed the expected enzymological properties: a NAD+-dependent ssa-oxidizing activity for DmSSADH and a NAD+-dependent acetaldehyde-oxidizing activity for Dm-ALDH. Site-directed mutageneses on DmSSADH performed in this study demonstrated that two residues essential for catalysis are glutamate 277 and cysteine 311. The second part of the thesis encompassed the gene identification, full length gene cloning of SSADH in Lc (LcSSADH) and Cf (CfSSADH), as well as functional expression of one gene version, respectively. Substrate/cosubstrate specificity determinations combined with enzyme kinetics studies showed that both enzymes are predominantly NAD+-dependent SSADHs. Bioinformatics analyses detected N-terminal mitochondrial import sequences in both Lc and Cf SSADH suggesting that these enzymes are localized in vivo in the mitochondrial matrix. The investigation of the genomic structure of the LcSSADH and CfSSADH genes revealed significant differences to the previously known gene organizations: firstly, different to the single copy SSADH gene situation in Ec, Dm, Mus musculus and Homo sapiens, Lc appears to possess 2-3 SSADH genes, while in Cf up to 8 gene copies may be present. Secondly, compared to Dm (2 exons, 1 intron), the exon/intron structure of the SSADH genes in Lc and Cf is not conserved: the one SSADH genomic gene version investigated in detail in Lc contained 3 exons and 2 introns, while the genomic gene version of Cf analysed in this study was devoid of any introns. The central topic of the third part of this thesis was the gene identification and biochemical characterization of SSADH from the tick Rm. By a combination of different PCR methods a tick gene orthologous to insect and mammalian SSADH (RmSSADH) could be identified and isolated. The results of Southern blot analyses of Rm DNA are incompatible with a single copy situation and suggest the presence of 2-3 RmSSADH genes. To compare RmSSADH with a mammalian SSADH, the respective gene was isolated from mouse. Both the tick and the mouse SSADH genes were then expressed as soluble functional proteins in Ec. The initial comparison showed that both proteins are potent NAD+-dependent ssa-oxidizing enzymes with very similar enzyme kinetics. A more detailed comparison of both enzymes suggests that in general, the mouse enzyme appears to be more specific for succinic semialdehyde than the tick enzyme. In the fourth section of the experimental part of this thesis, saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR) experiments were performed on three of the above SSADH preparations, to answer questions on the aldehyde substrate and cosubstrate binding to these enzymes. Importantly, the long-standing question whether the free aldehyde of succinic semialdehyde or its hydrated gem-diol form (present in aqueous solution in equimolar amounts) is the binding substrates was answered conclusively in favour of the aldehyde form for both the Ec and the Dm enzyme. Most remarkably, STD-NMR experimental investigation of the ssa interaction with the Dm SSADH cysteine311alanine mutant enzyme demonstrated binding of both the aldehyde and the gem-diol form. This experiment strongly suggests that cysteine311 is acting as an aldehyde versus gem-diol selectivity filter in the active site of the enzyme. Furthermore, STD-NMR epitope mapping of the NAD+/NADP+ binding to Ec SSADH and Dm SSADH was performed. In both cases, the data suggested that the dominant protein-ligand interactions are via the adenine and the nicotinamide ring systems, while the ribose moieties interact much less intensely with the enzymes.

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