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The elimination of endocrine disruptors, like bisphenol A, from wastewater is an environmental challenge of increasing importance, but no satisfying technical solution has been achieved until now. Thus, it was the aim of the present dissertation to increase the bisphenol A-affinity of yeast by cell surface-expression. Expressing a high affinity protein on its cell surface, the immobilised yeast could serve as a cheap feedstock in a bio filtration system for the elimination of bisphenol A traces from wastewater. Although, a cell own protein should be modified to increase its affinity and should be fused with an effectively anchor able cell wall protein, considering the cell proteome. In the present dissertation the “old yellow enzyme”, natively found in Saccharomyces cerevisiae, was modified by site-directed mutagenesis. A manual docking from the “old yellow enzyme” with bisphenol A was simulated and simulations of punctiform mutagenesis in the protein binding site were done based on the well known crystal structure of the protein. Five simulated mutagenesis products, which should deliver possible new or improved interactions between ligand and protein, were analysed with a secondary structure analysis program and with the Ramachandran plot. The chosen mutations were accomplished at the “old yellow enzyme”- gene by PCR and the belonging proteins were expressed in Escherichia coli. The mutated proteins were purified and investigated by equilibrium dialysis to compare the bisphenol A- binding properties and to determine the dissociation constant. The yield of pure protein was increased to a factor of 2.5 by optimising the induction conditions of Escherichia coli concerning incubation temperature and the amount of inducing substance. The reason for the increased yield was the decreased generation of inclusion bodies. An effective protein purification protocol has been developed, that delivered the required amount of 10 mg pure protein for the equilibrium dialysis with an acceptable effort of time and costs. Four of five investigated, purified proteins showing a mutation that should build an additional hydrogen bond to the beta-OH-group of bisphenol A, bound less bisphenol A than the wild type protein. Only the exchange of the amino acid on position 194, asparagine against aspartate, provoked an increase of the bisphenol A- affinity. This mutant protein had a five times higher affinity than the wild type “old yellow enzyme” and a determined Kd- value of 93 µM. The mutated gene N194D was coupled with three different genes of cell wall anchors from Saccharomyces cerevisiae, Aga, Cwp2 and Sed1, and was transformed into Saccharomyces cerevisiae by two different cell surface expression vectors, pYD and pFB. All fusion proteins were expressed on the cell surface and were localized by fluorescent antibodies. It was determined a significant increase of the bisphenol A binding speed for all modified strains following to cell surface expression of the fusion proteins. The maximum adsorbed bisphenol A amount was scarcely influenced by the expression of the fusion proteins. The strains based on the pFB- plamid, respectively the cell wall anchors Cwp2 and Sed1, were the most effective ones. Almost one minute after the beginning of incubation, 77% of their adsorption capacity, which was 475 respectively 510 µg bisphenol A per gram dry cell matter, was bound. In comparison to the wild type strain bound only 13%. The additional insertion of a 25 amino acid long glycine- serine- linker sequence between the cell wall anchor and the binding protein was ineffective. Less by the choice of the cell wall anchor but by the choice of the expression vector the strains based on the pFB- plasmids were determined as the most useful. The present dissertation delivers several new, optimized strains of Saccharomyces cerevisiae, which show a significant increased bisphenol A binding speed by expression of a cell own, punctiform mutated proteins on their cell surface. The strains show this behaviour under conditions of pH and temperature which are common in the effluent of wastewater treatment plants. The proteome of the cell was scarcely influenced by the genetic modification which could be an advantage for the permission for a technical use. The strains can now be used as a inexpensive feedstock for immobilization experiments which aim the construction of a bio filtration system to eliminate bisphenol A from wastewater.

One of the major problems associated with the use of antibiotics is the occurrence of antibiotic-related diarrhea. Although most such cases of diarrhea are harmless, in individual cases it may lead on to fulminant colitis. In 10-20% of the patients, the causal factor is infection with Clostridium difficile, which multiplies unhindered in patients on antibiotics. In the meantime, controlled, randomized studies have shown that probiotics can exercise a preventive effect in antibiotic-related diarrhea. The administration of Saccharomyces boulardii or Lactobacillus rhamnosus GG reduces the relative risk of an antibiotic-related diarrhea developing by 0.5. The data are more uniform in children than in adults.

GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnlines information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus , as well as curated information on 700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and SwissProt link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subjectoriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org . The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics. (VLID)2213581

0\. Titel und Inhalt I 1\. Einleitung 1 2\. Material und Methoden 16 3\. Ergebnisse 41 4\. Diskussion 85 5\. Zusammenfassung 99 6\. Abstract 101 7\. Literatur 103 8\. Anhang 121, Im Rahmen der in dieser Arbeit durchgeführten Two-Hybrid-Analysen wurden Pex7p, Pex14p und Pex8p als neue Interaktionspartner von Pex8p identifiziert. Interaktionen mit weiteren Peroxinen der Docking-Maschinerie, zu Peroxinen der Zinkfinger-Klasse und Peroxinen anderer Funktionskreise wurden nicht festgestellt. Der Interaktionsbereich zwischen Pex8p und Pex5p, Pex8p und Pex14p konnte auf die Aminosäuren 146-586 von Pex8p eingeengt werden. Der Interaktionsbereich zwischen Pex8p und Pex7p konnte nicht eingeengt werden. Pex7p benötigt zur Interaktion zu Pex8p das gesamte Pex8-Protein. Zur Interaktion mit Pex8p (146-586 aa) benötigt Pex5p die Aminosäuren 1-312. Die TPR-Domänen von Pex5p sind für die Interaktion zwischen Pex5p und Pex8p entbehrlich. Die Interaktion zwischen Pex8p und Pex14p wird sowohl von Peroxinen der Funktionskreise "Cargo-Rezeptor-Bindung", "Docking-Maschinerie" und "Rezeptor-Translokation" beeinflusst und ist daher indirekt. Die Interaktionen zwischen Pex8p und Pex5p sowie Pex8p und Pex7p werden durch kein Peroxin der untersuchten Funktionskreise vermittelt und scheinen daher direkt zu sein. Im Rahmen der Mutationsanalyse von Pex8p wurden drei Mutationen identifiziert, welche die Interaktion zwischen Pex8p und Pex5p, Pex7p und Pex14p auf unterschiedliche Weise unterbinden. Mutation Pex8pM-A(S231P) unterbindet die Interaktion zu Pex5p, Pex7p und Pex14p. Mutation Pex8pM-C(A238V) unterbindet die Interaktion zu Pex5p und Pex14p. Mutation Pex8pM-D(T327P) unterbindet die Interaktion zu Pex7p und Pex14p. Damit scheinen beide Rezeptoren für die Interaktion zwischen Pex8p und Pex14p notwendig zu sein. Die Pex8p-Deletionsmutante kann durch alle mutierten Pex8-Proteine komplementiert werden. Ebenfalls werden in diesen komplementierten pex8D-Zellen fluoreszenzmarkierte Cargoproteine in Peroxisomen importiert. Aufgrund der Mutationsanalysen von Pex8p ist es wahrscheinlich, dass Pex8p eine Lösung des Cargo-Rezeptor-Komplexes durch Konformationsänderung des Rezeptors bewirkt. Das Targeting von Pex8p zur peroxisomalen Membran in Abhängigkeit der PTS-Rezeptoren kann trotz der PTS- Signale innerhalb der Pex8p-Aminosäuresequenz ausgeschlossen werden. Dies wird durch Analysen zur Extrahierbarkeit des Proteins aus peroxisomalen Membranen unterstützt. Pex8p ist bei Abwesenheit von Pex5p für Proteinase K zugänglich. Somit beeinflußt Pex5p nicht das Targeting von Pex8p aber indirekt dessen Zugänglichkeit zu cytosolischen Komponenten., In the course of this study Pex7p, Pex14p and Pex8p were identified as new interaction partners of Pex8p, by means of two-hybrid-analysis. Interactions of further peroxines of the docking-machinery, to peroxines of the zincfinger- class and of other functional groups were not identified. The region of interaction of Pex8p and Pex5p, Pex8p and Pex14p was narrowed down to amino acids 146-586 of Pex8p. It was not possible to narrow down the region of interaction of Pex8p to Pex7p. Pex7p needs a full length Pex8-protein for interaction. For interaction with Pex8p (146-586 aa) Pex5p requires the amino acids 1-312. The TRP-domains of Pex5p are not required for the interaction between Pex5p and Pex8p. The interaction between Pex8p and Pex14p is influenced by peroxines of the "cargo-receptor-binding-group", the "docking- machinery-group", and the "receptor-translocation-group", and is therefore indirect. The interactions between Pex8p and Pex5p, as well as between Pex8p and Pex7p are not mediated by peroxines of the investigated functional groups and are therefore likely to be direct. By means of mutationanalysis of Pex8p, three mutations were identified, which prevent interactions between Pex8p and Pex5p, Pex7p and Pex14p in different ways. Mutation Pex8pM-A(S231P) prevents the interaction to Pex5p, Pex7p and Pex14p. Mutation Pex8pM-C(A238V) prevents the interaction to Pex5p and Pex14p. Mutation Pex8pM-D(T327P) prevents the interaction to Pex7p and Pex14p. Therefore, both receptors seem to be necessary for interaction between Pex8p and Pex14p. The Pex8p-mutant can be complemented by all mutated Pex8-proteins. Moreover, fluorescently labelled cargoproteins are imported into peroxisomes in complemented pex8D-cells. On the basis of the mutationanalysis of Pex8p it is likely, that Pex8p induces the release of the cargo-receptorcomplex due to changes of the receptor conformation. In spite of the presence of a PTS-signal in Pex8p, the targeting of Pex8p to the peroxisomal membrane is independent of the PTS-receptors. This is further supported by extraction analysis of the protein from peroxisomal membranes. In the absence of Pex5p, Pex8p can be degraded by Proteinase K. Therefore Pex5p does not influence the targeting of Pex8p but indirectly influences its approachability to the cytosolic components.

Pseudomembranous enterocolitis generally occurs after antibiotic treatment. The standard treatment is oral metronidazol or vancomycin. Nevertheless, relapses of Clostridium difficile enterocolitis are observed in 10-25% of cases. Factors associated with recurrences include endogenous reinfection by spore formation, selective IgG1 or IgA deficiency or infection with mutated strains of Clostridium difficile. Recurrent Clostridium difficile enterocolitis may be treated with repeat oral vancomycin combined with Sacchoromyces boulardii, with intravenous immunoglobulin for severe colitis.

Inducibility of alcohol dehydrogenase (ADH) crystals in yeast cells depends on the growth phase. They accumulate during exponential growth, a process that is parallelled by accumulation of a special protein (H protein). The following pieces of evidence show that crystals, H protein, and ADH are identical: (1) The three proteins have the same amino acid composition and subunit molecular weight; (2) active H protein and ADH exhibit the same electrophoretic mobility, indicating a common molecular weight of about 158 000 D; (3) the native H protein and a crystal fraction stabilized by Zn++ and dialyzed against EDTA possess ADH activity.

A method to prepare polyribosomes from yeasts by using the french-press is described. The highest yield of polyribosomes was derived from late log-phase cells. These polyribosomes, incubated in a cell-free system, were able to reinitiate protein synthesis, which was shown by inhibiting aminoacid incorporation by aurintricarboxylic acid, edeine and sodiumfluoride. We developed the translational system in order to look for the optimal ion-conditions of a DNA-dependent protein-synthesizing system. We found out that at the optimal MgCl2-concentration (6 mM) protein synthesis was strongly inhibited by Mangan ions which are required for transcription in yeast. If protein-synthesis was carried out with 2 mM and 3 mM MgCl2 maximal aminoacid incorporation was observed at 2 mM and 1.5 mM MnCl2.

Antimycotic (miconazole) medicated tampons were used for treatment in 53 patients with vaginal saccharomycetic infections as evidenced by hanging drop and on culture. A short term 2 1/2 day treatment with 5 tampons showed a success rate of 92.5%. The fast relief of symptoms like discharge, itching and burning is an improvement of the medicated tampon treatment. The new medicated tampons are considered as improvement of local vaginal treatment.

A purified fraction of crystals stabilized with Cd2+ is prepared from Saccharomyces carlsbergensis protoplasts by means of differential centrifugation. As proved by negative staining the crystals are hexagonal with well preserved surface which in its turn also reveals a hexagonal fine structure. Crystals are digestible by pronase (1 mg/m) in phosphate buffer (pH 7.4). The molecular weight of protein subunits is 38000+/-300D. Amino acid composition of the crude crystal protein is similar to that of microtubular protein (MTP) from brain and sperm, except that lysin content is much higher. The crystal protein exhibits more differences, however, after purification by preparative gel electrophoresis. The level of glycine, alamine, and valine is increased, whereas that of glutamic acid is decreased.

In 1973 one of the present authors described a new method of cytological observation based upon flattening of living cells. This method was tested with several microorganisms as bacteria, fungi and algae. The application of the method allows the clear resolution of cytological structures that are otherwise obscured or can hardly be recognized. In addition cellular motion and development can be followed much better than by usual methods of cell culture. In certain objects (yeasts and fungal hyphae tips) all cellular organelles are forced into one plane of focus and are thus resolved simultaneously even at highest magnification. Different species especially among the yeasts exhibited quite varying intracellular contrast of mitochondria.

The plant-pathogen fungus Trichothecium roseum produces in vitro as well as in vivo the toxic metabolites trichothecin, trichothecolone and rosenonolactone. Trichothecin is cytotoxic and inhibits the alcoholic fermentation. It is not metabolized by yeast during the alcoholic fermentation. All toxins showed a minor biological activity against microorganisms other than yeast. Trichothecin caused a bitter taste in wine at higher toxin concentrations (greater than 5 mg/l). Trichothecin was detected in a few samples of wine of higher quality.