TY  - JOUR
A1  - Gabellini, N.
A1  - Harnisch, U.
A1  - McCarthy, J. E.
A1  - Hauska, G.
A1  - Sebald, Walter
T1  - Cloning and expression of the fbc operon encoding the FeS protein, cytochrome b and cytochrome c\(_1\) from the Rhodopseudomonas sphaeroides b/c\(_1\) complex
N2  - The gene for the FeS protein of the Rhodopseudomonas sphaeroides b/c1 complex was identified by means of crosshybridization with a segment of the gene encoding the corresponding FeS protein of Neurospora crassa. Plasmids (pRSF1-14) containing the cross-hybridizing region, covering in total 13.5 kb of chromosomal DNA, were expressed in vitro in a homologous system. One RSF plasmid directed the synthesis of all three main polypeptides of the R. sphaeroides blc1 complex: the FeS protein, cytochrome b and cytochrome c1• The FeS protein and cytochrome c1 were apparently synthesized as precursor fonns. None of the pRSF plasmids directed the synthesis of the 10-kd polypeptide found in b/c1 complex preparations. Partial sequencing of the cloned region was performed. Several sites of strong homology between R. sphaeroides and eukaryotic polypeptides of the b/c1 complex were identified. The genes encode the three b/c1 polypeptides in the order: (5') FeS protein, cytochrome b, cytochrome c1• The three genes are transcribed to give a polycistronic mRNA of 2.9 kb. This transcriptional unit has been designated the jbc operon; its coding capacity corresponds to the size of the polycistronic mRNA assuming that only the genes for the FeS protein (jbcF), cytochrome b (jbcß) and cytochrome c1 (jbcC) are present. This could indicate that these three subunits constitute the minimal catalytic unit of the b/c1 complex from photosynthetic membranes.
KW  - Biochemie
KW  - R. sphaeroidesl
KW  - b/c1 complex
KW  - gene
KW  - cloning
KW  - in vitro expression
KW  - polycistronic mRNA
Y1  - 1985
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62642
ER  - 
TY  - JOUR
A1  - McCarthy, J. E.
A1  - Schairer, H. U.
A1  - Sebald, Walter
T1  - Translational initiation frequency of atp genes from Escherichia coli: identification of an intercistronic sequence that enhances translation
N2  - The c, b and ö subunit genes of the Escherichia coli atp operon were cloned individually in an expression vector between the tac fusion promoter and the galK gene. The relative rates of subunit synthesis directed by the cloned genes were similar in vitro andin vivo and compared favourably with the subunit stoichiometry of the assembled proton-translocating A TP synthase of E. coli in vivo. The rate of synthesis of subunit c was at least six times that of subunit b and 18 times that of subunit ö. Progressive shortening of the long intercistronic sequence lying upstream of the subunit c gene showed that maximal expression of this gene is dependent upon the presence of a sequence stretching > 20 bp upstream of the Shine-Dalgarno site. This sequence thus acts to enhance the rate of translational initiation. The possibility that similar sequences might perform the same function in other operons of E. coli and bacteriophage A is also discussed. Translation of the subunit b cistron is partially coupled to translation of the preceding subunit c cistron. In conclusion, the expression of all the atp operon genes could be adjusted to accommodate the subunit requirements of A TP synthase assembly primarily by means of mechanisms which control the efficiency of translational initiation and re-initiation at the respective cistron start codons.
KW  - Biochemie
KW  - E. coli atp operon
KW  - subunit stoichiometry
KW  - in vitro and in vivo expression
KW  - translational initiation
Y1  - 1985
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62657
ER  - 
TY  - JOUR
A1  - Lindenmaier, W.
A1  - Dittmar, K. E.
A1  - Hauser, H.
A1  - Necker, A.
A1  - Sebald, Walter
T1  - Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo
N2  - A method has been developed that allows the isolation of genomic clones from a cosmid library by homologaus recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene. The genomic cosmid library was packaged in vivo into A. phage particles. A recombination-proficient host strain carrying IL2 cDNA sequences in a non-homologaus plasmid vector was infected by the packaged cosmid library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both vectors were selected. From a recombinant cosmid clone the chromosomal IL2 genewas restored. After DNA mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively.
KW  - Biochemie
KW  - Recombinant DNA
KW  - DNA mediated gene transfer
KW  - expression plasmid
KW  - screening
KW  - packaging
KW  - bacteriophage lambda
Y1  - 1985
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62662
ER  - 
TY  - JOUR
A1  - Schmidt, B.
A1  - Wachter, E.
A1  - Sebald, Walter
A1  - Neupert, W.
T1  - Processing peptidase of Neurospora mitochondria. Two-step cleavage of imported ATPase subunit 9
N2  - Subunit 9 (dicyclohexylcarbodümide binding protein, 'proteolipid') of the mitochondrial F 1F0-ATPase is a nuclearly coded protein in Neurospora crassa. lt is synthesized on free cytoplasmic ribosomes as a larger precursor with an NH2-terminal peptide extension. The peptide extension is cleaved ofT after transport of the protein into the mitochondria. A processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and characterized using a cell-free system. Precursor synthesized in vitro was incubated with extracts of mitochondria. Processing peptidase required Mn2 + for its activity. Localization studies suggested that it is a soluble component of the mitochondrial matrix. The precursor was cleaved in two sequential steps via an intermediate-sized polypeptide. The intermediate form in the processing of subunit 9 was also seen in vivo and upon import of the precursor into isolated mitochondria in vitro. The two dcavage sites in the precursor molecule were determined. The data indicate that: {a) the correct NH2-terminus of the mature protein was generated, (b) the NH2-terminal amino acid of the intermediate-sized polypeptide is isoleueine in position -31. The cleavage sites show similarity ofprimary structure. It is concluded that processing peptidase removes the peptide extension from the precursor to subunit 9 (and probably other precursors) after translocation of these polypeptides (or the NHrterminal part of these polypeptides) into the matrix space of mitochondria.
KW  - Biochemie
Y1  - 1984
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62674
ER  - 
TY  - JOUR
A1  - Hoppe, J.
A1  - Schairer, H. U.
A1  - Sebald, Walter
T1  - The proteolipid of a mutant ATPase from Escherichia coli defective in H\(^+\)-conduction contains a glycine instead of the carbodiimide-reactive aspartyl residue
N2  - No abstract available
KW  - Biochemie
Y1  - 1980
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62769
ER  - 
TY  - JOUR
A1  - Sebald, Walter
A1  - Wachter, E.
A1  - Tzagoloff, A.
T1  - Identification of amino acid substitutions in the dicyclohexylcarbodiimide-binding subunit of the mitochondrial ATPase complex from oligomycin-resistant mutants of Saccharomyces cerevisiae
N2  - No abstract available
KW  - Biochemie
Y1  - 1979
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62770
ER  - 
TY  - THES
A1  - Alcantarino Menescal, Luciana
T1  - In vivo characterization of genetic factors involved in Xmrk driven melanoma formation in Medaka (Oryzias latipes): a closer look at braf, Stat5 and c-myc
T1  - In vivo Charakterisierung genetischer Faktoren mit Einfluss auf Xmrk induzierte Melanome in Medaka (Oryzias latipes): Untersuchung von braf, Stat5 und c-myc.
N2  - Melanoma arises from the malignant transformation of melanocytes and is one of the most aggressive forms of human cancer. In fish of the genus Xiphophorus, melanoma development, although very rarely, happens spontaneously in nature and can be induced by interspecific crossing. The oncogenic receptor tyrosine kinase, Xmrk, is responsible for melanoma formation in these fishes. Since Xiphophorus are live-bearing fishes and therefore not compatible with embryonic manipulation and transgenesis, the Xmrk melanoma model was brought to the medaka (Oryzias latipes) system. Xmrk expression under the control of the pigment cell specific mitf promoter leads to melanoma formation with 100% penetrance in medaka. Xmrk is an orthologue of the human epidermal growth factor receptor (EGFR) and activates several downstream signaling pathways. Examples of these pathways are the direct phosphorylation of BRAF and Stat5, as well as the enhanced transcription of C-myc. BRAF is a serine-threonine kinase which is found mutated at high frequencies in malignant melanomas. Stat5 is a transcription factor known to be constitutively activated in fish melanoma. C-myc is a transcription factor that is thought to regulate the expression of approximately 15% of all human genes and is involved in cancer progression of a large number of different tumors. To gain new in vivo information on candidate factors known to be involved in melanoma progression, I identified and analysed BRAF, Stat5 and C-myc in the laboratory fish model system medaka. BRAF protein motifs are highly conserved among vertebrates and the results of this work indicate that its function in the MAPK signaling is maintained in medaka. Transgenic medaka lines carrying a constitutive active version of BRAF (V614E) showed more pigmented skin when compared to wild type. Also, some transiently expressing BRAF V614E fishes showed a disrupted eye phenotype. In addition, I was able to identify two Stat5 copies in medaka, named Stat5ab/a and Stat5ab/b. Sequence analysis revealed a higher similarity between both Stat5 sequences when compared to either human Stat5a or Stat5b. This suggests that the two Stat5 copies in medaka arose by an independent duplication processes. I cloned these two Stat5 present in medaka, produced constitutive active and dominant negative gene versions and successfully established transgenic lines carrying each version under the control of the MITF promoter. These lines will help to elucidate questions that are still remaining in Stat5 biology and its function in melanoma progression, like the role of Stat5 phosphorylation on tumor invasiveness. In a third project during my PhD work, I analysed medaka C-myc function and indentified two copies of this gene in medaka, named c-myc17 and c-myc20, according to the chromosome where they are located. I produced conditional transgenic medaka lines carrying the c-myc17 gene coupled to the hormone binding domain of the estrogen receptor to enable specific transgene activation at a given time point. Comparable to human C-myc, medaka C-myc17 is able to induce proliferation and apoptosis in vivo after induction. Besides that, C-myc17 long-term activation led to liver hyperplasia. In summary, the medaka models generated in this work will be important to bring new in vivo information on genes involved in cancer development. Also, the generated transgenic lines can be easily crossed to the melanoma developing Xmrk medaka lines, thereby opening up the possibility to investigate their function in melanoma progression. Besides that, the generated medaka fishes make it possible to follow the whole development of melanocytes, since the embryos are transparent and can be used for high throughput chemical screens.
N2  - Melanome entstehen durch die krankhafte Transformation von Melanozyten und sind eine der aggressivsten Krebsarten beim Menschen. In Fischen der Gattung Xiphophorus können, wenn auch sehr selten, spontan Melanome entstehen oder durch spezielle Artenkreuzungen induziert werden. Grundlage für das Entstehen der Melanome in diesen Fischen ist die Rezeptortyrosinkinase Xmrk. Da alle Xiphophorus-Arten lebendgebärend sind und keine Manipulationen an Embryonen vorgenommen werden können, wurde ein Xmrk Melanommodel für Medaka (Oryzias latipes) etabliert. Die Expression von Xmrk in Pigmentzellen dieser Fischart resultiert mit 100%iger Penetranz in Melanomen. Das Xmrk ist ein Ortholog des menschlichen „epidermal growth factor“ (EGFR) und aktiviert verschiedene nachgeschaltete Signalwege. Beispiele für diese Aktivierungen sind die Phosphorylierung von BRAF, Stat5 und die erhöhte Expression von c-myc. BRAF ist eine Serin-Threoninkinase, welche oft in malignen Melanomen mutiert ist. Stat5 ist ein Transkriptionsfaktor, welcher dauerhaft in Fischtumoren aktiviert ist. C-myc ist ein Transkriptionsfaktor, welcher etwa 15% aller menschlichen Gene sowie die Entstehung vieler menschlicher Tumore reguliert. Um neue Einsichten in die Funktion der Kanidatengene im Prozess der Melanomentstehung in vivo zu erlangen, habe ich Orthologe von BRAF, Stat5 und C-myc bei Medaka identifiziert und analysiert. Die Domänen des BRAF Proteins sind hoch konserviert in allen Vertebraten. Weiterhin deuten die Ergebnisse meiner Arbeit auf eine Beibehaltung der Funktionen im MAPK Signalweg hin. Transgene Medakalinien, welche eine dauerhaft aktive Version des BRAF Gens (V614E) exprimieren, weisen einerseits eine stärkere Hautpigmentierung auf. Weiterhin treten in diesen Fischen Veränderungen der Augen auf. In einem weiteren Projekt meiner Arbeit gelang es mir, zwei Kopien des Stat5 Gens im Medaka zu identifizieren, Stat5ab/a und Stat5ab/b. Sequenzanalysen zeigten eine höhere Übereinstimmung zwischen den beiden Genkopien, als zwischen denen von Medaka und Menschen. Dieses Ergebnis deutet darauf hin, dass die beiden Medaka Gene durch eine unabhängige Duplikation entstanden. In meiner Arbeit habe ich beide Gene des Medakas kloniert und jeweils eine konstitutiv aktive und eine dominant negative Version der Gene hergestellt. Weiterhin konnte ich erfolgreich für jede Genversion eine transgene Medakalinie etablieren, welche die verschiedenen Genvarianten unter der Kontrolle des pigmentzellspezifischen Promoters des mitf Gens exprimieren. Diese Linien werden in Zukunft helfen, den Einfluss von Stat5 Signalen auf den Prozess der Melanomverbreitung und dessen Invasivität zu erklären. In einem dritten Projekt meiner Doktorarbeit untersuchte ich das Vorkommen und die Funktion der C-myc Gene des Medakas. Ich konnte zwei Genkopien identifizieren, c-myc17 und c-myc20, welche auf unterschiedlichen Chromosomen lokalisiert sind. Ich konnte induzierbare, stabil transgene Linien herstellen, welche ein Fusionsprotein aus C-myc17 und der Hormonbindungsdomäne des Östrogenrezeptors von Maus exprimiert. Diese Linie ermöglichte eine induzierbare Aktivität des Transgens. Vergleichbar zum menschlichen MYC ist C-myc17 fähig, nach Aktivierung Proliferation und Apoptose in vivo auszulösen. Dauerhafte Aktivierung über einen längeren Zeitraum führt in diesen Linien zu Hyperplasie in Leber. Die verschiedenen Fischmodelle, die während dieser Arbeit generiert wurden, werden essentiell sein, um neue Einsichten in die Rolle diese Faktoren während der Krebsentwicklung in vivo zu erlangen. Weiterhin ermöglichen diese transgenen Linien durch einfaches Auskreuzen auf Xmrk Linien, deren Einfluss auf die Verbreitung von Melanomen zu untersuchen. Letztendlich sind mit diesen Linien auch Untersuchungen der Entwicklung von Pigmentzellen über Zeit möglich, da die Embryonen transparent sind und sich für chemisches Hochdurchsatz-Screening eignen.
KW  - Japankärpfling
KW  - Melanom
KW  - Myc
KW  - Molekulargenetik
KW  - melanoma
KW  - medaka
KW  - BRAF
KW  - Stat5
KW  - c-myc
KW  - melanoma
KW  - medaka
KW  - BRAF
KW  - Stat5
KW  - c-myc
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-70762
ER  - 
TY  - JOUR
A1  - Kraeussling, Michael
A1  - Wagner, Toni Ulrich
A1  - Schartl, Manfred
T1  - Highly Asynchronous and Asymmetric Cleavage Divisions Accompany Early Transcriptional Activity in Pre-Blastula Medaka Embryos
N2  - In the initial phase of development of fish embryos, a prominent and critical event is the midblastula transition (MBT). Before MBT cell cycle is rapid, highly synchronous and zygotic gene transcription is turned off. Only during MBT the cell cycle desynchronizes and transcription is activated. Multiple mechanisms, primarily the nucleocytoplasmic ratio, are supposed to control MBT activation. Unexpectedly, we find in the small teleost fish medaka (Oryzias latipes) that at very early stages, well before midblastula, cell division becomes asynchronous and cell volumes diverge. Furthermore, zygotic transcription is extensively activated already after the 64-cell stage. Thus, at least in medaka, the transition from maternal to zygotic transcription is uncoupled from the midblastula stage and not solely controlled by the nucleocytoplasmic ratio.
KW  - Fische
KW  - Embryo
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68906
ER  - 
TY  - JOUR
A1  - Michel, R.
A1  - Wachter, E.
A1  - Sebald, Walter
T1  - Synthesis of a larger precursor for the proteolipid subunit of the mitochondrial ATPase complex of Neurospora crassa in a cell-free wheat germ system
N2  - No abstract available
KW  - Biochemie
Y1  - 1979
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62789
ER  - 
TY  - JOUR
A1  - Sebald, Walter
A1  - Graf, T.
A1  - Lukins, H. B.
T1  - The dicyclohexylcarbodiimide-binding protein of the mitochondrial ATPase complex from Neurospora crassa and Saccharomyces cerevisiae. Identification and isolation
N2  - Incubation of mitochondria from Neuraspara crassa and Saccharomyces cerevisiae with the radioactive ATPase inhibitor [14C]dicyclohexylcarbodiimide results in the irreversible and rather specific labelling of a low-molecular-weight polypeptide. This dicyclohexylcarbodiimide-binding protein is identical with the smallest subunit (Mr 8000) of the mitochondrial ATPase complex, and it occurs as oligomer, probably as hexamer, in the enzyme protein. The dicyclohexylcarbodiimide-binding protein is extracted from whole mitochondria with neutral chloroformjmethanol both in the free and in the inhibitor-modified form. In Neuraspara and yeast, this extraction is highly selective and the protein is obtained in homogeneaus form when the mitochondria have been prewashed with certain organic solvents. The bound dicyclohexylcarbodiimide Iabel is enriched in the purified protein up to 50-fold compared to whole mitochondria. Based on the amino acid analysis, the dicyclohexylcarbodiimide-binding protein from Neurospora and yeast consists of at least 81 and 76 residues, respectively. The content of hydrophobic residues is extremely high. Histidine and tryptophan are absent. The N-terminal ~mino acid is tyrosine in Neuraspara and formylmethionine in yeast.
KW  - Biochemie
Y1  - 1979
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62792
ER  - 
TY  - JOUR
A1  - Graf, T.
A1  - Sebald, Walter
T1  - The dicyclohexylcarbodiimide-binding protein of the mitochondrial ATPase complex from beef heart. Isolation and amino acid composition
N2  - No abstract available
KW  - Biochemie
Y1  - 1978
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62806
ER  - 
TY  - JOUR
A1  - Jackl, G.
A1  - Sebald, Walter
T1  - Identification of two products of mitochondrial protein synthesis associated with mitochondrial adenosine triphosphatase from Neurospora crassa
N2  - Soluble mitochondrial ATPase (F1) isolated from Neurospora crassa is resolved by dodecylsulfate- gel electrophoresis into five polypeptide bands with apparent molecular weights of 59000, 55000, 36000, 15000 and 12000. At least nine further polypeptides remain associated with ATPase after disintegration of mitochondria with Triton X-100 as shown by the analysis of an immunoprecipitate obtained with antiserum to F 1 A TPase. Two of the associated polypeptides with apparent molecular weights of 19000 and 11000 are translated on mitochondrial ribosomes, as demonstrated by incorporation in vivo of radioactive leueine in the presence of specific inhibitors of mitochondrial (chloramphenicol) and extramitochondrial ( cycloheximide) protein synthesis. The appearance of mitochondrial translation products in the immunoprecipitated A TPase complex is inhibited by' cycloheximide. The same applies for some of the extramitochondrial translation products in the presence of chloramphenicol. This suggests that both types of polypeptides are necessary for the assembly of the A TPase complex.
KW  - Biochemie
Y1  - 1975
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62812
ER  - 
TY  - JOUR
A1  - Chipperfield, Joseph D.
A1  - Dytham, Calvin
A1  - Hovestadt, Thomas
T1  - An Updated Algorithm for the Generation of Neutral Landscapes by Spectral Synthesis
N2  - Background: Patterns that arise from an ecological process can be driven as much from the landscape over which the process is run as it is by some intrinsic properties of the process itself. The disentanglement of these effects is aided if it possible to run models of the process over artificial landscapes with controllable spatial properties. A number of different methods for the generation of so-called ‘neutral landscapes’ have been developed to provide just such a tool. Of these methods, a particular class that simulate fractional Brownian motion have shown particular promise. The existing methods of simulating fractional Brownian motion suffer from a number of problems however: they are often not easily generalisable to an arbitrary number of dimensions and produce outputs that can exhibit some undesirable artefacts. Methodology: We describe here an updated algorithm for the generation of neutral landscapes by fractional Brownian motion that do not display such undesirable properties. Using Monte Carlo simulation we assess the anisotropic properties of landscapes generated using the new algorithm described in this paper and compare it against a popular benchmark algorithm. Conclusion/Significance: The results show that the existing algorithm creates landscapes with values strongly correlated in the diagonal direction and that the new algorithm presented here corrects this artefact. A number of extensions of the algorithm described here are also highlighted: we describe how the algorithm can be employed to generate landscapes that display different properties in different dimensions and how they can be combined with an environmental gradient to produce landscapes that combine environmental variation at the local and macro scales.
KW  - Landschaft
KW  - Monte-Carlo-Simulation
KW  - Brownsche Bewegung
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68938
ER  - 
TY  - THES
A1  - Kronhardt, Angelika
T1  - Channel Formation, Binding and Translocation Properties of Anthrax, CDT and Related Toxins of the AB7 type
T1  - Kanalbilidung, Bindungs- und Translokationseigenschaften des Anthrax, CDT und verwandten Toxinen des AB7-Toxintyps
N2  - The ability to produce toxins is spread among a huge variety of bacterial strains. A very prominent class of bacterial protein toxins is the family of binary AB toxins sharing a common mode of intoxication. A pore forming component B binds and translocates an enzymatic component A into the cytosol of target cells exhibiting a fatal mode of action. These components are supposed to be not toxic themselves but both required for cell toxicity. Anthrax toxin produced by the Gram-positive bacteria Bacillus anthracis is the best studied binary toxin especially since its use as a biological weapon in the context of the attacks of 9/11 in 2001. In contrast to other binary toxins, Anthrax toxin possesses two different enzymatic components, edema factor (EF), a calcium- and calmodulin-dependent adenylat-cyclase and lethal factor (LF), a zinc-dependent metalloprotease. Protective antigen (PA) is the pore-forming component responsible for binding and translocation. Clostridium botulinum possesses in addition to the well known botulinum toxin (Botox) a variety of other toxins, such as the binary C2 toxin. C2 toxin is composed of the binding and translocation moiety C2II and the enzymatic moiety C2I acting as an actin-ADP-ribosyltransferase. In this study, the mode of translocation and the binding kinetics to the enzymatic component were studied in a biophysical experimental setup. In chapter 2, the binding of the N-terminal fractions EFN and LFN to the PA channel are analyzed in artificial bilayer membranes revealing lower binding affinity compared to full-length EF and LF. Other biophysical properties like voltage-dependency and ionic-strength dependency are not influenced. The results suggest that additional forces are involved in the binding process, than those concerning the N-terminus exclusively, as it was supposed previously. As the treatment of an Anthrax infection with antibiotics is often medicated very late due to the lack of early symptoms, tools to prevent intoxication are required. 4-aminoquinolones like chloroquine are known to block the PA channel, thereby inhibiting intoxication but they also lead to severe side-effects. In chapter 3 new promising agents are described that bind to PA in artificial bilayer systems, elucidating common motives and features which are necessary for binding to PA in general. The possible interaction of Anthrax and C2 toxin is investigated by measuring the binding of one enzymatic component to the respective other toxin’s pore (chapter 4). Interestingly, in vitro experiments using the black lipid bilayer assay show that PA is able to bind to C2I resulting in half saturation constants in the nanomolar range. Furthermore, in vivo this combination of toxin components exhibits cell toxicity in human cell lines. This is first-time evidence that a heterologous toxin combination is functional in in vitro and in vivo systems. In contrast, C2II is able to bind to EF as well as to LF in vitro, whereas in in vivo studies almost no toxic effect is detected. In the case of PA, an N-terminal His6-tag attached to the enzymatic subunit increased the binding affinity (chapter 5). A His6-tag attached to not related proteins also led to high binding affinities, providing the possibility to establish PA as a general cargo protein. In chapter 6 a set of different molecules and proteins is summarized, which are either related or not related to binary toxins, PA is able to bind. In first line, the presence of positive charges is found to be responsible for binding to PA which is in accordance to the fact that PA is highly cation selective. Furthermore, we present evidence that different cationic electrolytes serve as a binding partner to the PA channel. In the last decade another toxin has aroused public attention as it was found to be responsible for a rising number of nosocomial infections: Clostridium difficile CDT toxin. The mode of action of the enzymatic subunit CDTa is similar to C2I of C2 toxin, acting as an ADP-ribosylating toxin. The channel forming and binding properties of CDT toxin are studied in artificial bilayer membranes (chapter 7). We found that two different types of channels are formed by the B component CDTb. The first channel is similar to that of iota toxin’s Ib of Clostridium perfringens with comparable single channel conductance, selectivity and binding properties to the enzymatic subunit CDTa. The formation of this type of channel is cholesterol-dependent, whereas in the absence of cholesterol another kind of channel is observed. This channel has a single channel conductance which is rather high compared to all other binary toxin channels known so far, it is anion selective and does not show any binding affinity to the enzymatic component CDTa. The results reveal completely new insights in channel formation properties and the flexibility of a pore-forming component. Additionally, these findings suggest further possibilities of toxicity of the pore forming component itself which is not known for any other binary toxin yet. Therefore, the pathogenic role of this feature has to be studied in detail.
N2  - Die Fähigkeit, Toxine zu produzieren, ist unter verschiedensten Bakterienstämmen sehr verbreitet. Zu diesen Toxinen zählt auch die Familie der binären AB-Toxine, die hauptsächlich von Bakterien der Gattung Bacillus und Clostridium gebildet werden. Charakteristisch für diese bakteriellen Proteintoxine ist der Wirkungsmechanismus der Zellintoxikation. Eine porenformende Untereinheit B bindet eine enzymatische Untereinheit A und transportiert diese in das Zytosol von Zielzellen, die dort tödliche Wirkung entfalten. Es wird angenommen, dass die einzelnen Komponenten an sich nicht toxisch sind, sondern nur in Kombination Zellvergiftung auslösen. Anthrax-Toxin, das von dem Gram-positiven Bakterium Bacillus anthracis produziert wird, ist das bekannteste und am besten untersuchte binäre Toxin, besonders seit es im Jahr 2001 als Biowaffe eingesetzt wurde. Im Gegensatz zu anderen binären Toxinen besitzt das Anthrax-Toxin zwei enzymatische Komponenten: Edema Factor (EF), eine kalzium- und calmodulinabhängige Adenylatzyklase, und Lethal Factor (LF), eine zinkabhängige Metalloprotease. Protective Antigen (PA) ist die porenformende Komponente, die für die Binding und die Translokation der enzymatischen Untereinheiten verantwortlich ist. Clostridium botulinum produziert neben dem bekannten Botulinumtoxin (Botox) eine Reihe weiterer Toxine, unter anderem das binäre C2 Toxin. Dieses besteht aus der Binde- und Translokationskomponente C2II und der enzymatischen Komponente C2I, die als ADP-Ribosyltransferase fungiert. Im Rahmen der vorliegenden Arbeit werden der Translokationsmechanismus und die kinetischen Bindeeigenschaften dieser Toxine biophysikalisch untersucht. In Kapitel 2 wird die Bindung der N-terminalen Fragmente EFN und LFN an den PA-Kanal in künstlichen Lipidmembranen analysiert. Obwohl die Spannungs- und Ionenstärkeabhängigkeit unverändert sind, weisen die verkürzten Proteine deutlich geringe Bindeaffinitäten zu PA im Vergleich zu den vollständigen Proteinen auf. Die Ergebnisse zeigen, dass, anders als bisher angenommen, weitere Kräfte als die zwischen dem N-Terminus und dem PA-Kanal eine Rolle für die Bindung der enzymatischen Komponente spielen. Da bei einer Anthraxinfektion häufig keine frühen Symptome sichtbar sind, erfolgt die Behandlung mit Antibiotika in der Regel relativ spät. Daher werden neue Wirkstoffe benötigt, um einer Intoxikation vorzubeugen. Es ist bekannt, dass 4-Aminoquinolone, wie zum Beispiel Chloroquin, in der Lage sind, die PA-Pore zu blockieren und somit eine Zellvergiftung zu verhindern, allerdings haben diese Wirkstoffe starke Nebenwirkungen. In Kapitel 3 werden neue, vielversprechende Wirkstoffe beschrieben, die an PA binden können und Aufklärung darüber geben, welche Eigenschaften für die Bindung an PA im Allgemeinen verantwortlich sind. Des Weiteren wird untersucht, ob eine Kreuzreaktion zwischen den Komponenten des Anthrax- und C2-Toxins möglich ist (Kapitel 4). Dazu wird die Bindung einer enzymatischen Komponente an die Pore des entsprechenden anderen Toxins gemessen. Interessanterweise ergeben in vitro Experimente an künstlichen Lipidmembranen, dass PA an C2I bindet und in vivo Vergiftungen an humanen Zelllinien auslöst. Damit wird zum ersten Mal gezeigt, dass eine heterologe Toxinkombination sowohl in vitro als auch in vivo funktionell ist. C2II hingegen ist zwar in der Lage, EF und LF zu binden, die Transportrate in Zielzellen ist jedoch sehr gering. Im Fall von PA bewirkt ein N-terminaler His6-tag, der an die enzymtischen Einheiten gekoppelt ist, eine Erhöhung der Bindeaffinität, beschrieben in Kapitel 5. Dies ist sowohl für nah verwandte Proteine der Fall als auch für Proteine, die nicht im Zusammenhang mit binären Toxinen stehen. Somit eröffnet sich die Möglichkeit, PA als universelles Transportprotein zu nutzen. In Kapitel 6 werden verschiedene Moleküle und Proteine beschrieben, die in der Lage sind, an PA zu binden. Vor allem positive Ladungen scheinen für die Bindung an PA-Kanäle verantwortlich zu sein, was mit der Tatsache, dass PA stark kationenselektiv ist, im Einklang steht. Des Weiteren wird zum ersten Mal beschrieben, dass verschiedene Kationen selbst als Bindepartner fungieren können. Seit einigen Jahren ist ein weiteres Toxin in den Fokus der Öffentlichkeit gerückt, da es zunehmend für nosokomiale Infektionen verantwortlich gemacht wird: CDT-Toxin von Clostridium difficile. Wie das C2-Toxin besitzt CDT-Toxin ADP-Ribosyltransferaseaktivität, was zu irreversiblen Schäden des Aktin- Zytoskeletts und somit zum Zelltod führt. Die biophysikalischen Eigenschaften, betreffend Porenbildung und Bindeaffinität des CDT-Toxins werden in Kapitel 7 beschrieben. Wir zeigen, dass die B Komponente CDTb fähig ist, zwei unterschiedliche Kanäle zu bilden. Einer dieser Kanäle ist dem des Iota-Toxins von Clostridium perfringens ähnlich, die Einzelkanalleitfähigkeit, Selektivität und Bindeeigenschaften sind vergleichbar. Die Bildung dieses Kanals ist abhängig von Cholesterin, wohingegen in Abwesenheit von Cholesterin überwiegend ein anderer Kanal geformt wird. Dieser zeigt eine für einen binären Toxinkanal ungewöhnlich hohe Einzelkanalleitfähigkeit, der Kanal ist anionselektiv und weist keinerlei Bindeaffinität zu der enzymatischen Komponente CDTa auf. Die Ergebnisse offenbaren neue Einblicke in die Formierung von Toxinkanälen und deuten darauf hin, dass dieses Toxin durch die Flexibilität der Kanalbildung möglicherweise zusätzliche Fähigkeiten besitzt, Zellintoxikation auszulösen. Dennoch ist die physiologische und pathogene Rolle dieser Eigenschaft noch weitestgehend ungeklärt und bedarf intensiver Untersuchung.
KW  - Bacillus anthracis
KW  - Toxin
KW  - Lipidmembran
KW  - Pore
KW  - Translokation
KW  - Porenbildung
KW  - Anthrax toxin
KW  - Black lipid bilayer
KW  - pore formation and translocation
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-71559
ER  - 
TY  - THES
A1  - Cook, Mandy
T1  - The neurodegenerative Drosophila melanogaster AMPK mutant loechrig
T1  - The neurodegenerative Drosophila melanogaster AMPK Mutante loechrig
N2  - In dieser Doktorarbeit wird die Drosophila Mutante loechrig (loe), die progressive Degeneration des Nervensystems aufweist, weiter beschrieben. In der loe Mutante fehlt eine neuronale Isoform der γ- Untereinheit der Proteinkinase AMPK (AMP-activated protein kinase). Die heterotrimere AMPK (auch als SNF4Aγ bekannt) kontrolliert das Energieniveau der Zelle, was ständiges Beobachten des ATP/AMP- Verhältnis erfordert. AMPK wird durch niedrige Energiekonzentrationen und Beeinträchtigungen im Metabolismus, wie zum Beispiel Sauerstoffmangel, aktiviert und reguliert mehrere wichtige Signaltransduktionswege, die den Zellmetabolismus kontrollieren. Jedoch ist die Rolle von AMPK im neuronalen Überleben noch unklar. Eines der Proteine, dass von AMPK reguliert wird, ist HMGR (hydroxymethylglutaryl-CoA- reductase), ein Schlüsselenzym in der Cholesterin- und Isoprenoidsynthese. Es wurde gezeigt, dass wenn die Konzentration von HMGR manipuliert wird, auch der Schweregrad des neurodegenerativen Phänotyps in loe beeinflusst wird. Obwohl die regulatorische Rolle von AMPK auf HMGR in Drosophila konserviert ist, können Insekten Cholesterin nicht de novo synthetisieren. Dennoch ist der Syntheseweg von Isoprenoiden zwischen Vertebraten und Insekten evolutionär konserviert. Isoprenylierung von Proteinen, wie zum Beispiel von kleinen G-Proteinen, stellt den Proteinen einen hydophobischen Anker bereit, mit denen sie sich an die Zellmembran binden können, was in anschließender Aktivierung resultieren kann. In dieser Doktorarbeit wird gezeigt, dass die loe Mutation die Prenylierung von Rho1 und den LIM-Kinasesignalweg beeinflusst, was eine wichtige Rolle im Umsatz von Aktin und axonalem Auswachsen spielt. Die Ergebnisse weisen darauf hin, dass die Mutation in LOE, Hyperaktivität des Isoprenoidsynthesewegs verursacht, was zur erhöhten Farnesylierung von Rho1 und einer dementsprechend höheren Konzentration von Phospho- Cofilin führt. Eine Mutation in Rho1 verbessert den neurodegenerativen Phänotyp und die Lebenserwartung von loe. Der Anstieg vom inaktiven Cofilin in loe führt zu einer Zunahme von filamentösen Aktin. Aktin ist am Auswachen von Neuronen beteiligt und Experimente in denen loe Neurone analysiert wurden, gaben wertvolle Einblicke in eine mögliche Rolle die AMPK, und dementsprechend Aktin, im Neuronenwachstum spielt. Des Weiteren wurde demonstriert, dass Neurone, die von der loe Mutante stamen, einen verlangsamten axonalen Transport aufweisen, was darauf hinweist dass Veränderungen, die durch den Einfluss von loe auf den Rho1 Signalweg im Zytoskelettnetzwerk hervorgerufen wurden, zur Störung des axonalen Transports und anschließenden neuronalen Tod führen. Es zeigte außerdem, dass Aktin nicht nur am neuronalen Auswachsen beteiligt ist, sondern auch wichtig für die Aufrechterhaltung von Neuronen ist. Das bedeutet, dass Änderungen der Aktindynamik zur progressiven Degeneration von Neuronen führen kann. Zusammenfassend unterstreichen diese Ergebnisse die wichtige Bedeutung von AMPK in den Funktionen und im Überleben von Neuronen und eröffnen einen neuartigen funktionellen Mechanismus in dem Änderungen in AMPK neuronale Degeneration hervorrufen kann.
N2  - In this thesis the Drosophila mutant loechrig (loe), that shows progressive degeneration of the nervous system, is further described. Loe is missing a neuronal isoform of the protein kinase AMPK γ subunit (AMP-activated protein kinase- also known as SNF4Aγ) The heterotrimeric AMPK controls the energy level of the cell, which requires constant monitoring of the ATP/AMP levels. It is activated by low energy levels and metabolic insults like oxygen starvation and regulates multiple important signal pathways that control cell metabolism. Still, its role in neuronal survival is unclear. One of AMPK’s downstream targets is HMGR (hydroxymethylglutaryl-CoA- reductase), a key enzyme in cholesterol and isoprenoid synthesis. It has been shown that manipulating the levels of HMGR affects the severity of the neurodegenerative phenotype in loe. Whereas the regulatory role of AMPK on HMGR is conserved in Drosophila, insects cannot synthesize cholesterol de novo. However, the synthesis of isoprenoids is a pathway that is evolutionarily conserved between vertebrates and insects. Isoprenylation of target proteins like small G-proteins provides a hydrophobic anchor that allows the association of these proteins with membranes and following activation. This thesis shows that the loe mutation interferes with the prenylation of Rho1 and the regulation of the LIM kinase pathway, which plays an important role in actin turnover and axonal outgrowth. The results suggest that the mutation in LOE, causes hyperactivity of the isoprenoid synthesis pathway, which leads to increased farnesylation of RHO1 and therefore higher levels of phospho-cofilin. A mutation in Rho1 improves the neurodegenerative phenotype and life span. The increased inactive cofilin amount in loe leads to an up regulation of filamentous actin. Actin is involved in neuronal outgrowth and experiments analyzing loe neurons gave valuable insights into a possible role of AMPK and accordingly actin on neurite growth and stability. It was demonstrated that neurons derived from loe mutants exhibit reduces axonal transport suggesting that changes in the cytoskeletal network caused by the effect of loe on the Rho1 pathway lead to disruptions in axonal transport and subsequent neuronal death. It also shows that actin is not only involved in neuronal outgrowth, its also important in maintenance of neurons, suggesting that interference with actin dynamics leads to progressive degeneration of neurons. Together, these results further support the importance of AMPK in neuronal function and survival and provide a novel functional mechanisms how alterations in AMPK can cause neuronal degeneration
KW  - Taufliege
KW  - Nervendegeneration
KW  - AMP
KW  - Proteinkinasen
KW  - Molekulargenetik
KW  - Drosophila
KW  - Neurodegeneration
KW  - AMPK
KW  - Drosophila
KW  - Neurodegeneration
KW  - Rho
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-72027
ER  - 
TY  - JOUR
A1  - Krauss, Jochen
A1  - Gallenberger, Iris
A1  - Steffan-Dewenter, Ingolf
T1  - Decreased Functional Diversity and Biological Pest Control in Conventional Compared to Organic Crop Fields
N2  - Organic farming is one of the most successful agri-environmental schemes, as humans benefit from high quality food, farmers from higher prices for their products and it often successfully protects biodiversity. However there is little knowledge if organic farming also increases ecosystem services like pest control. We assessed 30 triticale fields (15 organic vs. 15 conventional) and recorded vascular plants, pollinators, aphids and their predators. Further, five conventional fields which were treated with insecticides were compared with 10 non-treated conventional fields. Organic fields had five times higher plant species richness and about twenty times higher pollinator species richness compared to conventional fields. Abundance of pollinators was even more than one-hundred times higher on organic fields. In contrast, the abundance of cereal aphids was five times lower in organic fields, while predator abundances were three times higher and predator-prey ratios twenty times higher in organic fields, indicating a significantly higher potential for biological pest control in organic fields. Insecticide treatment in conventional fields had only a short-term effect on aphid densities while later in the season aphid abundances were even higher and predator abundances lower in treated compared to untreated conventional fields. Our data indicate that insecticide treatment kept aphid predators at low abundances throughout the season, thereby significantly reducing top-down control of aphid populations. Plant and pollinator species richness as well as predator abundances and predator-prey ratios were higher at field edges compared to field centres, highlighting the importance of field edges for ecosystem services. In conclusion organic farming increases biodiversity, including important functional groups like plants, pollinators and predators which enhance natural pest control. Preventative insecticide application in conventional fields has only short-term effects on aphid densities but long-term negative effects on biological pest control. Therefore conventional farmers should restrict insecticide applications to situations where thresholds for pest densities are reached.
KW  - Landwirtschaft
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69005
ER  - 
TY  - JOUR
A1  - Leonhardt, Sara D.
A1  - Schmitt, Thomas
A1  - Blüthgen, Nico
T1  - Tree Resin Composition, Collection Behavior and Selective Filters Shape Chemical Profiles of Tropical Bees (Apidae: Meliponini)
N2  - The diversity of species is striking, but can be far exceeded by the chemical diversity of compounds collected, produced or used by them. Here, we relate the specificity of plant-consumer interactions to chemical diversity applying a comparative network analysis to both levels. Chemical diversity was explored for interactions between tropical stingless bees and plant resins, which bees collect for nest construction and to deter predators and microbes. Resins also function as an environmental source for terpenes that serve as appeasement allomones and protection against predators when accumulated on the bees’ body surfaces. To unravel the origin of the bees’ complex chemical profiles, we investigated resin collection and the processing of resin-derived terpenes. We therefore analyzed chemical networks of tree resins, foraging networks of resin collecting bees, and their acquired chemical networks. We revealed that 113 terpenes in nests of six bee species and 83 on their body surfaces comprised a subset of the 1,117 compounds found in resins from seven tree species. Sesquiterpenes were the most variable class of terpenes. Albeit widely present in tree resins, they were only found on the body surface of some species, but entirely lacking in others. Moreover, whereas the nest profile of Tetragonula melanocephala contained sesquiterpenes, its surface profile did not. Stingless bees showed a generalized collecting behavior among resin sources, and only a hitherto undescribed species-specific ‘‘filtering’’ of resin-derived terpenes can explain the variation in chemical profiles of nests and body surfaces fromdifferent species. The tight relationship between bees and tree resins of a large variety of species elucidates why the bees’ surfaces contain a much higher chemodiversity than other hymenopterans.
KW  - Stachellose Biene
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69035
ER  - 
TY  - JOUR
A1  - Brandstätter, Andreas
A1  - Rössler, W.
A1  - Kleineidam, C. J.
T1  - Friends and foes from an ant brain's point of view - neuronal correlates of colony odors in a social insect
N2  - Background: Successful cooperation depends on reliable identification of friends and foes. Social insects discriminate colony members (nestmates/friends) from foreign workers (non-nestmates/foes) by colony-specific, multi-component colony odors. Traditionally, complex processing in the brain has been regarded as crucial for colony recognition. Odor information is represented as spatial patterns of activity and processed in the primary olfactory neuropile, the antennal lobe (AL) of insects, which is analogous to the vertebrate olfactory bulb. Correlative evidence indicates that the spatial activity patterns reflect odor-quality, i.e., how an odor is perceived. For colony odors, alternatively, a sensory filter in the peripheral nervous system was suggested, causing specific anosmia to nestmate colony odors. Here, we investigate neuronal correlates of colony odors in the brain of a social insect to directly test whether they are anosmic to nestmate colony odors and whether spatial activity patterns in the AL can predict how odor qualities like ‘‘friend’’ and ‘‘foe’’ are attributed to colony odors. Methodology/Principal Findings: Using ant dummies that mimic natural conditions, we presented colony odors and investigated their neuronal representation in the ant Camponotus floridanus. Nestmate and non-nestmate colony odors elicited neuronal activity: In the periphery, we recorded sensory responses of olfactory receptor neurons (electroantennography), and in the brain, we measured colony odor specific spatial activity patterns in the AL (calcium imaging). Surprisingly, upon repeated stimulation with the same colony odor, spatial activity patterns were variable, and as variable as activity patterns elicited by different colony odors. Conclusions: Ants are not anosmic to nestmate colony odors. However, spatial activity patterns in the AL alone do not provide sufficient information for colony odor discrimination and this finding challenges the current notion of how odor quality is coded. Our result illustrates the enormous challenge for the nervous system to classify multi-component odors and indicates that other neuronal parameters, e.g., precise timing of neuronal activity, are likely necessary for attribution of odor quality to multi-component odors.
KW  - Ameisen
KW  - Geruch
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69046
ER  - 
TY  - JOUR
A1  - Albrecht, Marco
A1  - Sharma, Cynthia M.
A1  - Dittrich, Marcus T.
A1  - Müller, Tobias
A1  - Reinhardt, Richard
A1  - Vogel, Jörg
A1  - Rudel, Thomas
T1  - The Transcriptional Landscape of Chlamydia pneumoniae
N2  - Background: Gene function analysis of the obligate intracellular bacterium Chlamydia pneumoniae is hampered by the facts that this organism is inaccessible to genetic manipulations and not cultivable outside the host. The genomes of several strains have been sequenced; however, very little information is available on the gene structure and transcriptome of C. pneumoniae. Results: Using a differential RNA-sequencing approach with specific enrichment of primary transcripts, we defined the transcriptome of purified elementary bodies and reticulate bodies of C. pneumoniae strain CWL-029; 565 transcriptional start sites of annotated genes and novel transcripts were mapped. Analysis of adjacent genes for cotranscription revealed 246 polycistronic transcripts. In total, a distinct transcription start site or an affiliation to an operon could be assigned to 862 out of 1,074 annotated protein coding genes. Semi-quantitative analysis of mapped cDNA reads revealed significant differences for 288 genes in the RNA levels of genes isolated from elementary bodies and reticulate bodies. We have identified and in part confirmed 75 novel putative non-coding RNAs. The detailed map of transcription start sites at single nucleotide resolution allowed for the first time a comprehensive and saturating analysis of promoter consensus sequences in Chlamydia. Conclusions: The precise transcriptional landscape as a complement to the genome sequence will provide new insights into the organization, control and function of genes. Novel non-coding RNAs and identified common promoter motifs will help to understand gene regulation of this important human pathogen.
KW  - Chlamydia pneumoniae
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69116
ER  - 
TY  - THES
A1  - Gätschenberger, Heike
T1  - Die Expression humoraler und zellulärer Immunreaktionen bei Drohnenlarven und adulten Drohnen der Honigbiene (Apis mellifera)
T1  - Expression of humoral and cellular immune reactions of dronelarvae and adult drones of the honey bee (Apis mellifera)
N2  - Soziale Insekten wie die Honigbiene (Apis mellifera) besitzen ein breites Spektrum an Abwehrmechanismen gegen Pathogenbefall, sowohl auf der Ebene der Kolonie (soziale Immunität) als auch auf der Stufe des Individuums (angeborenes Immunsystem). Die Hauptaufgabe der relativ kurzlebigen Drohnen besteht in der Begattung von Jungköniginnen. Daher stellte sich die Frage, ob auch die Drohnen ähnlich den Arbeiterinnen mit energieaufwendigen Immunreaktionen auf Infektionen reagieren. Wie im Folgenden beschrieben, konnte ich nachweisen, dass Drohnen eine ausgeprägte Immunkompetenz besitzen. Das angeborene Immunsystem setzt sich aus humoralen und zellulären Abwehrreaktionen zusammen. Bei der humoralen Immunantwort werden bestimmte evolutionär konservierte Signalkaskaden aktiviert, an deren Ende die Expression einer Vielzahl von antimikrobiellen Peptiden (AMPs) und immunspezifischen Proteinen (IRPs) steht. Zur Analyse der humoralen Immunantwort wurden von mir zum einen Hemmhoftests durchgeführt, um die gesamte antimikrobielle Aktivität der Haemolymphe nach artifizieller Infektion zu ermitteln und zum anderen spezifische AMPs bzw. IRPs identifiziert. Hierzu wurden die Haemolymphproteine in ein- oder zwei-dimensionalen Polyacrylamidgelen aufgetrennt und ausgewählte Proteinbanden bzw. -spots mittels nano HPLC/Massenspektrometrie analysiert. Die Hauptkomponenten des zellulären Immunsystems sind Wundheilung, Phagozytose, Einkapselung und Nodulation. In meiner Arbeit habe ich zum ersten Mal Noduli bei infizierten Drohnen nachweisen können. Frisch geschlüpfte adulte Drohnen (1d) weisen ein breites Spektrum an Immunreaktionen auf, das sowohl humorale als auch zelluläre Immunantworten umfasst. Nach Infektion mit dem Gram-negativen Bakterium E.coli und verschiedenen bakteriellen Zellwandbestandteilen wie Lipopolysaccharid (LPS), Peptidoglycan (PGN) und 1,3ß-Glucan (Bestandteil von Pilzzellwänden), werden die AMPs Hymenoptaecin, Defensin 1 und Abaecin induziert. Desweiteren exprimieren junge adulte Drohnen eine Reihe hochmolekularer immunspezifischer Proteine (IRPs) wie z.B. Carboxylesterase (CE 1), eine Serinprotease, die möglicherweise an der Prozessierung der Prophenoloxidase beteiligt ist, ein Peptidoglycan-interagierendes Protein (PGRP-S2) und zwei Proteine unbekannter Funktion, IRp42 und IRp30. Parallel zu bekannten bienenspezifischen AMPs wurde ein animales Peptidtoxin (APT) in Drohnenlarven, adulten Drohnen und adulten Hummeln nach E.coli Infektion in der Haemolymphe nachgewiesen. Von dem als OCLP 1 (ω-conotoxin-like protein 1) benannten Peptid war bereits bekannt, dass es in Fischen paralytische und damit toxische Effekte auslöst. Meine Beobachtungen lassen vermuten, dass es sich bei OCLP 1 um ein Peptidtoxin mit antimikrobiellen Eigenschaften und damit um eine neue Klasse von AMPs handelt. Die allgemeine humorale Immunkompetenz scheint während der gesamten Lebensspanne adulter Drohnen (~ 7 Wochen) konstant zu bleiben, wie durch die gleichbleibende antimikrobielle Aktivität im Hemmhoftest gezeigt wurde. Junge Drohnen reagieren auf eine E.coli Infektion mit der Bildung zahlreicher Noduli (~1000 Noduli/Drohn), die vor allem entlang des Herzschlauches zu finden sind. Diese zelluläre Immunantwort nimmt mit dem Alter der Drohnen ab, so dass bei 18 d alten Drohnen nur noch rund 10 Noduli/Drohn gefunden werden. Auf der anderen Seite nimmt die phagozytotische Aktivität bei älteren Drohnen scheinbar zu. In einer Reihe von parallel laufenden Versuchsreihen konnte ich eindrucksvoll zeigen, dass zelluläre Immunreaktionen wie Phagozytose und Nodulation unmittelbar nach bakterieller Infektion einsetzen. Hierbei erreicht die Nodulibildung 8-10 h p.i. eine Plateauphase, wohingegen die humorale Immunantwort erst 6 h p.i. schwach einsetzt, danach stetig zunimmt und noch 72 h p.i. nachweisbar ist. Es ist mir gelungen, eine Methode zur künstlichen Aufzucht von Drohnenlarven zu etablieren. Diese ermöglichte konstante und sterile Versuchsbedingungen zur Untersuchung der Immunreaktionen von Larven. Nach Infektion mit E.coli reagieren Drohnenlarven mit einer starken Aktivierung ihrer humoralen Immunantwort durch die Expression von AMPs, jedoch werden keine hochmolekularen IRPs wie in adulten Drohnen hochreguliert. Zudem ist die Nodulibildung in Larven nur schwach ausgeprägt. Völlig unerwartete Beobachtungen wurden beim Studium der Immunkompetenz von Drohnenpuppen gemacht. Nach Injektion lebender E.coli Zellen in Drohnenpuppen stellte ich eine dramatische Veränderung im Aussehen der Puppen fest. Die Puppen verfärbten sich gräulich schwarz. Genauere Untersuchungen haben dann gezeigt, dass die Drohnenpuppen, wie auch die der Arbeiterinnen, offensichtlich keine zelluläre Abwehrreaktion aktivieren können und die humorale Immunantwort nur sehr schwach ausfällt und viel zu spät einsetzt.
N2  - Social insects like honey bees (Apis mellifera) possess a wide range of defence mechanisms against pathogens on the colony level (social immunity) as well as on the individual level (innate immunity). In early summer, honey bee colonies consist of about 50.000 workers, a few hundred drones and one queen. The main task of the short-lived drones is to mate with a virgin queen. This raises a question: do drones, similar to workers mount an energy-intense immune reaction to fend off infections? In my thesis I could show that drones exhibit an effective immune competence. The innate immune response is composed of a humoral and a cellular component. In the humoral immune response, evolutionally conserved signalling pathways are activated and lead to the induced synthesis of antimicrobial peptides (AMPs) and immune responsive proteins (IRPs). In order to analyse the humoral immune response, I conducted inhibition zone assays as well as one- and two-dimensional gelelectrophoresis. Afterwards, HPLC/MS was performed in order to identify specific protein spots. Wound healing, phagocytosis, encapsulation and nodulation are the principal components of the cellular immune system. In my work, I could show nodulation reactions in infected drones for the first time. Newly emerged drones (1d) respond to infections with a wide range of immune reactions, including humoral and cellular defence mechanisms. The AMPs hymenoptaecin, defensin 1 and abaecin are induced after infection with gram-negative E.coli, bacterial cell wall components like lipopolysaccharides (LPS), peptidoglycan (PGN) and 1,3ß-glucan (cell wall component of fungi). In addition, young drones express some high molecular immune responsive proteins (IRPs) like carboxylesterase (CE 1), a serine protease, that is potentially part of the prophenoloxidase activating system, further a peptidoglycan recognition protein (PGRP-S2) and with IRp30 and IRp42 two proteins of unknown function. IRp42 possibly belongs to the glycin-rich proteins (GRP) because of its glycin–rich regions. Glycin-rich proteins are known to participate in host defence in plants. IRp30 is a leucine-rich repeat containing protein with a C-terminal leucine zipper, which is common among Hymenopterans. Therefore it is possible for IRp30 to interact with other proteins, like cell wall structures of pathogens. I detected the bee-specific lysozyme 2 in the haemolymph of adult drones after septic infection. It belongs to the chicken (c)-type lysozymes like lysozyme 1, which are potentially active against gram-positive and gram-negative bacteria and fungi in insects. After E.coli infection, an animal peptide toxin (APT) was detected simultaneously to the known AMPs in the haemolymph of larvae, adult drones and adult bumble bees. It is known that this peptide, called OCLP 1 (ω-conotoxin-like protein 1), triggers paralytic and thus toxic effects in fish. My observations suggest that OCLP 1 is probably a peptide toxin with antimicrobial characteristics, and thus could belong to a new class of AMPs. Throughout their whole life (~ 7 weeks), adult drones maintain their immune competence. This was shown by the continuous antimicrobial activity of drone haemolymph in inhibition zone assays. After E.coli infection, young drones react with nodule formation (~1000 noduli/drone), which are mostly attached to the dorsal vessel. With increasing age, this type of cellular immune response weakens, so that 18d old drones only produce 10 noduli/drone. On the other hand, the phagocytic activity seems to increase in older drones In an array of parallel tests, I showed the immediate onset of the cellular immune reactions phagocytosis and nodulation upon septic infections. Nodule formation reaches a plateau 8-10 h p.i., whereas humoral immune response just begins to start 6 h p.i., continuously rises and is still measurable 72 h p.i.. I succeeded in establishing a method for rearing honey bee drone larvae artificially. This enabled constant and sterile conditions for the testing of immune reactions in larvae. A strong activation of humoral immunity with the expression of AMPs resulted from E.coli infection, yet no immune responsive proteins were induced like in adult drones. Moreover, nodule formation in larvae is weak. While studying immune competence of drone pupae, I made a surprising observation. There is a dramatic change in the physical appearance of drone pupae after injecting living E.coli bacteria. They change colour to a greyish black. Precise examinations revealed that there is no cellular immune response in drone pupae as well as in worker pupae, only a weak humoral immune reaction which is initiated too late.
KW  - Humorale Immunität
KW  - Zelluläre Immunität
KW  - Drohne
KW  - Biene
KW  - Apis mellifera
KW  - Drohnen
KW  - humorales und zelluläres Immunsystem
KW  - Apsi mellifera
KW  - drones
KW  - humoral and cellular Immune reactions
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-71960
ER  -