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Mit zunehmender Industrialisierung der Lebensmittelproduktion werden Produkten vermehrt Wirkstoffe wie Farb-, Konservierungs- und Aromastoffe zugesetzt, um sie aufzuwerten. Hierbei wird von Konsumenten das Prädikat „natürlich“ bevorzugt, das beispielsweise durch die enzymatische Produktion dieser Wirkstoffe erhalten wird. Hier bieten sich Basidiomycota als Genspender an, da sie als Hauptdestruenten von Pflanzenresten ein breites jedoch bisher nur unzureichend beschriebenes Spektrum an potenten degradativen Enzymen bieten. In dieser Arbeit wurde an der natürlichen Produktion der Raucharomakomponente 4 Vinylguajakol (4 VG) sowie dem potentiellen Konservierungsstoff 2,6-Dimethoxychinon (DMBQ) durch degradative basidiomycetische Enzyme gearbeitet. In einer ersten Studie wurde eine Ferulasäuredecarboxylase aus Schizophyllum commune (ScoFAD) als erstes Enzym seiner Art aus Basidiomycota identifiziert und erfolgreich im lebensmittelgeeigneten Expressionswirt Komagataella phaffii heterolog produziert. Im Vergleich mit bakteriellen und ascomycetischen Enzymen zeigte die ScoFAD mit 0,16 mmol L 1 die höchste Affinität zu ihrem einzigen Substrat Ferulasäure. Die katalytische Effizienz übertraf die homologer Enzyme mit 4.779 L s-1 mmol-1 um mehr als den Faktor 50. Die ScoFAD wurde kovalent auf AminoLink Plus Agarose immobilisiert und zeigte in einem kontinuierlichen Prozess über mehrere Tage Aktivität. So wurde mit der Synthese von natürlichem 4-VG als Schlüsselkomponente des Raucharomas der Weg für neue, nachhaltige und sichere Raucharomen geebnet. DMBQ besitzt antibakterielle und antifungale Eigenschaften und wurde in vorangegangenen Arbeiten als in-vitro-Oxidationsprodukt von Malvidin nachgewiesen. In einer zweiten Studie wurden erstmals komparative Analysen des DMBQ-Gehalts verschiedener Weine durchgeführt. Das Chinon wurde lediglich in den malvidinhaltigen Proben mit Konzentrationen zwischen 28 und 137 µg L 1 nachgewiesen, was auf das Vorhandensein des gleichen Bildungsweges in vivo hindeutete. Es konnten erstmals in-vivo-Bildungskinetiken in homogenisierten roten Früchten aufgenommen werden, die eine Zelldekompartementalisierung als Auslöser der DMBQ-Bildung zeigten. Zudem wurde ein Bioprozess etabliert, der die Produktion des Chinons unter food-grade-Bedingungen mit Ausbeuten von 94 % und 65 mg DMBQ/100 g Weintrester mittels einer Laccase aus Pleurotus pulmonarius ermöglicht., The growing industrialisation of food production goes hand in hand with the increasing addition of active compounds such as dyes, preservatives, and flavours to improve the products’ properties. In such additives, consumers prefer the classification “natural”, which can be achieved by enzymatically catalysed synthesis. A suitable source of such enzymes are destruents like Basidiomycota, as they produce a broad variety of potent but not well described degrading enzymes. This work was focussed on the natural production of the smoke flavour compound 4 vinylguaiacol (4-VG) and the potential preservative 2,6-dimethoxybenzoquinone (DMBQ) via degrading basidiomycetous enzymes. In a first study, a ferulic acid decarboxylase from Schizophyllum commune (ScoFAD) was successfully identified as the first of its kind in Basidiomycota and was heterologously produced in the food-grade host Komagataella phaffii. In contrast to homologous bacterial and ascomycetous enzymes, ScoFAD offered the highest described affinity of 0.16 mmol L 1 towards its only substrate, ferulic acid. Further, its catalytic efficiency of 4,779 L s 1 mmol-1 exceeded the next best known enzyme by more than a factor of 50. The enzyme was covalently immobilised on AminoLink Plus Agarose and showed activity for several days in a continuous process. This enzymatic synthesis of natural 4-VG as a key compound of smoke paves the way for a new generation of sustainable and safe smoke flavours. The compound DMBQ offers anti-bacterial and anti-fungal activities and was identified as an oxidation product of malvidin in previous studies. In a second study, comparative analysis of the DMBQ content of different wine beverages were performed for the first time. The quinone was solely found in malvidin containing wines with concentrations ranging from 28 to 137 µg L-1, indicating that DMBQ formation also takes place in vivo. Formation kinetics in anthocyanin-rich fruits were recorded and demonstrated that quinone formation is triggered by cell decompartmentalisation. Further, a bioprocess was established enabling the production of natural DMBQ from wine pomace via a laccase of Pleurotus pulmonarius with yields of 94 % and 65 mg DMBQ/100 g wine pomace.

4207 Bei der vorliegenden Dissertation handelt es sich um die erste Forschungsarbeit zur inositolbasierten Signaltransduktion im Basidiomyzeten S. commune. Des Weiteren befasst sich die Arbeit mit der Rolle der Inositolmonophosphatase bei der Signaltransduktion. Durch die Verbindung von In-silico-Analyse mit experimentellen Laboruntersuchungen konnten wichtige Erkenntnisse zur zukünftigen Beschreibung der inositolbasierten Signaltransduktion gewonnen werden. Die mutmaßliche Wechselwirkung zwischen der Inositolmonophosphatase und Ras1 konnte im Rahmen dieser Forschungsarbeit mittels RT-qPCR bestetätigt werden. Dies suggeriert, dass eine Verbindung zwischen dem Inositolmonophosphatase und Ras1 existiert, wobei die genauen Mechanismen noch unbekannt sind. Im Rahmen dieser Arbeit wurde erstmalig die Inositolmonophosphatase aus S. commune extrahiert und mittels Strong Acid Exchange-High Liquid Performance Chromatography (SAX-HPLC) analysiert. Das Elutionsprofil zeigte, dass S. commune Inositolmonophosphatase (IP1), Inositoldiphosphat (IP2), Inositoltriphosphat (IP3), Inositoltetrakisphosphat (IP4), Inositolpentakisphosphat (IP5), Inositolhexakisphosphat (IP6), Diphosphoinositol-Pentakisphosphat (IP7) und Bis-Diphosphoinositol-Tetrakisphosphat (IP8) verstoffwechseln kann. Lithium, ein Inhibitor der Inositolmonophosphatase, verursachte eine Änderung des gesamten Inositolphosphatprofils und induzierte die Produktion „höherer“ Inositolphosphatspezies wie IP3, IP4, IP5, IP6 und die energiereichen Inositolpyrophosphat-Signalmoleküle (IP7 und IP8). Die Induktion von hohen Inositphosphaten durch Lithium zeigt einen molekularen Komplex von Inositolphosphatkinasen an. Lithium veränderte allerdings nicht das Inositolphosphatprofil der aktiven Ras1-Mutante, was darauf schließen lässt, dass die Aktivierung von Ras1 ggf. die inhibitorische Wirkung von Lithium auf die Inositolmonophosphatase aufheben kann, und somit der Hyperphosphorylierung hoher Inositolphosphatspezies entgegenwirkt. Lithium führte zu einer signifikaten Verringerung auf das Pilzwachstum im Wildtyp, was auf das veränderte Inositolphosphatprofil zurückgeführt werden kann. Dieses Phänomen wurde jedoch nicht in der Ras1-Mutante beobachtet. Des Weiteren inhibierte Lithium die Entwicklung und Bildung von Fruchtkörpern bei S. commune; es gab jedoch keine Auswirkung auf den Kreuzungsvorgang und die Schnallenbildung. Die Verstoffwechselung von Inositolphosphaten ist von großer Bedeutung für die Entwicklung des Pilzes. Die Synthese von Inositolphosphaten wird durch die regulierte Aktivität metabolisierender Enzyme wie Phosphatasen und Kinasen kontrolliert. Für die Entwicklung des von monokaryotischen und dikaryotischen werden IP6 und die Inositolpyrophosphate IP7 und IP8 benötigt, wohingegen die IP6, IP7, und IP8 im Fruchtkörper reduziert sind. Die Proteom-Analyse zeigte wichtige Informationen über die Regulation von Inositol-basierten Signalmolekülen. Die Histidinsäurephosphatase- Domäne der Inositolpolyphosphat-Phosphatasen, das Enzym, welches Inositphosphat dephosphoryliert, wurde in einer geringeren Häufigkeit im Fruchtkörper gefunden, so wie auch die Myoinositol-1-Phosphat-Synthase und Zusammenfassung 94 die Phosphatidylinositol-3- und -4-Kinase. Die Verringerung der Inositolphosphat- und Phosphatidylinositol-Signalproteine zeigt, dass beide Signalkaskaden nicht aktiviert sind, wenn der Fruchtkörper ausgereift ist. Andererseits zeigt sich, dass die Überexpression der Inositolmonophosphatase in S. commune eine wichtige Rolle in der Regulierung von Transportprozessen innerhalb der Zelle spielt. Proteom-Analysen zeigten die Induktion mehrerer Proteine, die am Zelltransport beteiligt sind, sowie die Induktion motorischer Zytoskelettproteine, die am Transport von Vesikeln und Vakuolen entlang der Hyphe mitwirken. Dementsprechend kann die Inositolmonophosphatase mit dem Wachstum und der Polarisierung der Pilzhyphe assoziiert werden. Mittels Laserscanning-Mikroskopie wurde die Morphologie von membranhaltigen Organellen in Mutanten mit imp-Überexpression im Vergleich zu einer Kontrollen beobachtet und es konnte gezeigt werden, dass große Vakuolen in imp-Überexpressionsmutanten häufiger vorhanden sind.

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Der Einsatz von Enzymen in Waschmittelformulierungen steigt seit Jahrzehnten beständig an. Zu den am meist verwendeten Enzymen gehören die α-Amylasen, wobei deren bakterieller Ursprung überwiegt. Zudem sind Pilze in der Lage eine Vielfalt an extrazellulären Enzymen zu bilden, um sich ihren Lebensraum zu erschließen. In der vorliegenden Arbeit wurden neuartige pilzliche α-Amylasen für den Einsatz in Waschmittelformulierungen gesucht. Insgesamt wurden dabei über 50 verschiedene Basidiomycota kultiviert und deren Sekretom nach waschaktiven α-Amylasen gescreent. Dafür wurden sowohl Aktivitätsassays als auch Applikationsstudien durchgeführt. Es stellte sich heraus, dass mit Rhizoctonia solani, Trametes hirsuta, Irpex lacteus, Fomes fomentarius, Fomitopsis pinicola, Pleurotus sajor-caju, Pycnoporus sanguineus und Lentinus strygosus acht Stämme α-Amylasen mit einer verbesserten Waschleistung produzierten. Es gelang dabei nach einer Reinigung mittels IEX und anschließender Analyse mittels SDS-PAGE sowie Zymografien, die waschaktiven Enzyme zu separieren und mittels Massenspektrometrie zu identifizieren. Anhand der durchgeführten de-novo Sequenzierung konnten die α-Amylasen von R. solani, T. hirsuta, I. lacteus, F. fomentarius, F. pinicola, und P. sanguineus identifiziert werden. Ausgehend von den korrespondierenden Genomdaten wurden Primer abgeleitet und schließlich die codierenden Gene amplifiziert. Bei einem Vergleich der α-Amylasen zeigte sich, dass Amylasen mit einer Stärke bindenden Domäne, der CBM20, die besten Waschleistungen im isoaktiven Vergleich erzielten. Von sechs identifizierten α-Amylasen weisen R. solani, T. hirsuta, F. fomentarius und F. pinicola diese Domäne auf, was verglichen mit der natürlichen Auftrittswahrscheinlichkeit von zehn Prozent eine Anhäufung zeigt. Der Versuch einige ausgewählte α-Amylase-Gene in Komagataella phaffii zu exprimieren, führte nicht zum gewünschten Erfolg, auch nicht mit Hilfe von molekularbiologischen Varianten, wie der Anpassung der codon usage, der Änderung der Signalsequenzen oder der Produktion als Fusionsprotein. Daher sollte mit Aspergillus oryzae ein neuer Expressionsstamm designt werden, bei welchem mittels der CRISPR-Technik die wirtseigenen Amylasen ausgeknockt werden sollten. Nach der Sequenzierung der Genombereiche, stellte sich heraus, dass es zu keinen CRISPR vermittelten Deletionen im Bereich der Zielgene gekommen war.

Introduction of durable resistance genes in crops is an important strategy to prevent yield loss caused by pathogens. The durable multi-pathogen resistance gene Lr34 originating from wheat is widely used in breeding, and is functionally transferable to barley and rice. The molecular resistance mechanism of Lr34, encoding for an adenosine triphosphate-binding cassette transporter, is not known yet. To understand the molecular function and the defense response of durable disease resistance in cereals, the metabolic response of Lr34 was investigated in, except for the Lr34 gene, genetically identical lines of barley, rice and wheat. A broad range of compounds including primary, secondary and lipophilic metabolites were analyzed by a combination of gas (GC) and liquid chromatography (LC) mass spectrometry (MS) based methods. Data from metabolomics correlated well with transcriptomics data for plant defense responses such as the formation of anti-fungal hordatines or the components of the glyoxylate cycle. Induction of the glyoxylate cycle found in transgenic Lr34 rice grown in the greenhouse was confirmed in field-grown natural Lr34 wheat. Constitutively active plant defense responses were observed in the different cereals.

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The majority of mycoses which lead to mycotic tumors in patients without any predisposing underlying disease are either caused by Cryptococcus gattii and C. neoformans or by dematiaceous fungi which include Cladophialophora bantiana, Ramichloridium mackenziei, Exophiala and Fonsecaea species. The detection of hyphae in granuloma in the brain should lead to screening for pigmented fungi, which are recognized best in hematoxylin eosin (HE) or sometimes also in periodic acid-Schiff (PAS) stained sections. In patients who survive a near drowning accident and those who develop brain abscesses, scedosporiosis should always be considered as a possible infection.

The present work shows the study on the elucidation of the biosynthetic pathway to sesquiterpenoid hybrid natural products, armillylorsellinates and melleolides, the class of natural products with the highest described structural diversity and interesting antimicrobial and cytotoxic activity. Due to the high complexity and diversity of synthesized molecules and the lack of possibilities to manipulate the natural producers Armillaria sp., there is great interest in elucidation of the biosynthetic pathway. This consists of cloning and functional characterization of involved genes and enzymes. After the attempted isolation of the key enzyme, the terpene cyclase protoilludene synthase to identify a homogeneous protein fraction, identification and cloning of the gene was achieved by sequencing of 2592 cDNA sequences of Armillaria gallica FU02472 transcriptome. The enzyme activity characterization of the native enzyme from A. gallica FU02472 and the heterologous protein expressed in E. coli as 6-Protoilludensynthase was achieved by the establishment of a radioactive activity assay and radio-thin-layer chromatography as well as a non - radioactive assay and detection of the biocatalysis products by GC/MS. Determining the copy number of the terpene cyclase showed that it was a single copy in the genome. Screening of a genomic library led to the identification of a 22.9 kb DNA fragment. Sequencing and comparison with the NCBI BLAST database confirmed the expected clustered organization of genes presumably associated to the biosynthesis. The following identification of an adjacent genomic fragment allowed the identification of four cytochrome P450 monooxygenases and two reading frames with previously uncharacterized function in addition to the terpene cyclase. The cloning of the cDNA sequences and the analysis of genomic clones showed a very complex structure of the coding (exon) and noncoding (intron) sequences for all the considered genes. With an average intron length of ~ 50 bp, for example, an exon was identified consisting of only five base pairs. This complex structure led to the cloning of incorrectly processed cDNA sequences. After correction and putative functional cloning, the four cytochrome P450 monooxygenases were subjected to functional testing of candidates with tritium-labeled 6-protoilludene, the exclusive product of the enzymatic reaction of farnesyl diphosphate catalyzed by Protoilludene synthase. For this purpose, in vivo feeding experiments with Saccharomyces cerevisiae (S. cerevisiae) were carried out and demonstrated the activity of the enzyme CYP Arm3. The construction of a plasmid-based S. cerevisiae production strain by expression of the catalytic domain of the HMG-CoA reductase, for the partial decoupling of feedback regulation mechanisms of the mevalonate pathway, NADPH:cytochrome P450 reductase for the cofactor regenerating, the identified terpene cyclase protoilludene synthase and P450 monooxygenase CYP Arm3 allowed the heterologous production of a putative monohydroxylated sesquiterpene skeleton. By isolation of the metabolite from the solvent extract of cell mass and culture supernatant, the position of hydroxylation was determined by NMR analysis. Based on the identification of 8alpha-hydroxy-6- Protoilludene another intermediate the 8alpha ,13-hydroxy- Protoilluden was identified using the established system. Functional characterization and heterologous expression of the terpene synthase and of two P450 monooxygenases shows for the first time the production of dihydroxylated sesquiterpene from higher fungi, in particular from basidiomycetes. This observation indicates that the biosynthetic pathway branches after the formation of terpene skeleton 6-protoilludene to the formation of armillylorsellinates and melleolides. The modification in the protoilludene skeleton starts with hydroxylation of the six-membered ring and modifications. Finally the transfer of the side chain (orsellinic acid - polyketide), according to the putative biosynthetic pathway, is catalyzed. The engineered strain is a platform development for the production of highly modified protoilludene skeletons for chemical or enzymatic semisynthesis to generate armillylorsellinate or melleolide derivatives for enhanced antibiotic and cytotoxic activity testing. The successful development of pharmaceutically relevant metabolites has been shown for example for illudin analogues (cytostatics) and pseudomutilines (terpene-based antibiotics).

The medicinal use of mushrooms, so-called higher fungi, has a very long tradition in the Asian countries, whereas their use in the Western hemisphere has been slightly increasing only since the last decades. The paper gives an overview about the most important medicinal mushrooms and summarizes the actual knowledge about chemistry and pharmacology of Lentinula edo-des (Shiitake, Golden Oak Mushroom), Ganoderma lucidum (Reishi, Ling Zhi), Agaricus brasiliensis (Royal sun agaricus), Grifola frondosa (Maitake, Hen-of-the-Woods) and Hericium erinaceus (Yamabushitake, Lion's Man, Monkey's Head).

There are many mycotoxins with different chemical structures and biological activities. They are present in many food items of vegetable and animal origin as well as in the air. Consequently the exposure of humans to these agents is rather common. In some particular instances the dosage taken up by the body may be considerable. The various biological effects of mycotoxins such as organ toxicity, mutagenicity, carcinogenicity, teratogenicity and modulation of the immune system are well documented by in vitro and animal experiments. Furthermore, cases of intoxication by mycotoxins are well known in veterinary medicine. However, the relevance of mycotoxins in human medicine remains largely underestimated, because material from patients with characteristic symptoms is rarely tested for mycotoxins.

The boron contents of 68 edible common macrofungi of Hungary were analysed. The average boron content of the examples was 11.74 mg/kg dry mass, this is higher than the average value of green plants of Hungary (7.4 mg/kg dry mass). Although the highest boron contents were found in the examples from woods near Budapest, the differences between the various sites are not significant. It seems that the site has not an important effect on the boron content of mushrooms. Some taxonomical groups are examples of higher, others are examples of lower boron content. The highest concentration of boron (54.3 mg/kg dry mass) was found in the species Marasmius wynnei which indicates a bioconcentration of four to five fold to the average boron level. Significant bioaccumulation of analysed edible species was not established, but the poisoning mushroom Mycena pura has a very great (401-607 mg/kg dry mass) boron concentration. This accumulation is observed to the first time.

Fifty six samples of edible, wide-spread species of higher fungi were analysed for copper-, manganese- and zinc-contents. The average copper concentration of the samples is high (56.16 mg/kg dry mass) as compared to the green plants. Some groups of fungi (Agaricus and Macrolepiota species) are considerable copper-accumulators (until 226 mg/kg DM). Pronounced accumulation of manganese was reported in the case of Lepista nebularis and Pluteus atricapillus. The zinc-content of fungi (average: 104.1 mg/kg DM, between 7.71 mg/kg (Lepista nebularis) and 194.3 mg/kg (Agaricus silvaticus) is relatively balanced, accumulation were not established.

The amino acid concentrations of six samples (five species) of the genus Russula and of seven samples (five species) of the genus Agaricus were determined. The Russula samples have significantly lower total amino acid concentrations (14.79% of the dry weight), than the Agaricus species (23.75%). Essential amino acids occur in both groups in appreciable concentrations: 48.9% of the total amino acid concentration for Russula and 46.4% for Agaricus. Between the cultivated varieties and the wild species of Agaricus are insignificant differences in the total amino acid concentration, but the quantities of certain essential amino acids were lower, whereas the concentration of Glu was higher in the wild species.

A comparative analysis of crude protein, crude ash, phosphorus (P), potassium (K) and calcium (Ca) contents of 57 samples of eight common edible mushroom species was made. The most important protein sources were: Marasmius oreades and Lepista nebularis. Species of the Boletaceae formed an intermediate group, while relatively proteinless species were: Armillariella mellea and Cratarellus cornucopioides. The lowest crude protein content was established in Cantharellus cibarius. The ash contents varied more widely. The greatest P contents were measured in Lepista nebularis and Marasmius oreades but most mushrooms contained 6-7 gP/kg. The analysed mushroom samples contained 30-40 gK/kg dry weight and 0,2-0,3 gCa/kg. These analyses are important from the point of view of the nutritional role of mushrooms.

46 species of 10 families of basidiomycetes were screened for trypsin-inhibitor activity. These mushrooms were extracted with water to estimate the inhibitor activity. High inhibitor activity (trypsin inhibition higher than 50%) was estimated in 8 species of 5 families.

Radioactive contamination of wild-growing mushrooms collected in the Northern Black Forest and neighbouring Rhine Valley area has been monitored since the Chernobyl reactor accident. Cs-134, CS-137, Ag-100m and the natural nuclide K-40 were determined by gamma-spectrometry. Pooled data from all species and all locations showed a decrease in the radio-caesium levels from 1986 to 1988, both in mean values and maximum values. Samples of the same species collected at the same location exhibited large differences, although mixed samples rather than individual mushrooms were measured. Accumulation of caesium was observed particularly in Laccaria amethystina, Xerocomus badius, Xerocomus chrysenteron, Cantharellus tubaeformis, Laccaria laccata, and Russula ochroleuca, with maximum values of 3600 (1986), 2000 (1987), and 1200 (1988) Bq/kg of radiocaesium. A large number of species, among them Calocybe gambosa and Coprinus comatus, showed very small accumulations or no caesium. An accumulation of silver was found in Macrolepiota rhacodes and Agaricus arvensis, which contained a maximum of 11 Bq/kg Ag-110m.

Oyster mushrooms are cultivated for human consumption in various subspecies. Their spores are a highly potent allergen, causing an exogenous allergic alveolitis. Relevant early pointers are the typical anamnesis, reduced vital capacity without any signs of obstruction, slight changes on x-ray film, positive immunofluorescence test. Frequent and typical wrong diagnoses during the initial episodes of the disease are: respiratory infection, influenza, pleuritis, type I allergy. So far, antibodies were identified in 76 immunofluorescence tests performed in 98 exposed subjects; 29 of these demonstrated signs of disease. Antibody formation is definitely subspecies-specific. Doubtful cases of "habituation" or negative antibody levels can be explained by a change in subspecies cultivation and hence a different antigen spectrum. The only therapeutically effective method is active prevention at the place of work as well as avoidance or elimination of the causative allergen. Drug treatment is useless. The only effective measure is to certify temporary disability to keep the patient away from the allergen.

With dopa as substrat the enzyme were incubated 20 min in 0.1 M phosphate buffer, pH 6.8. After that the activity was detected at wavelengths of 313 and 492 nm using a layer thickness of 10 mm. Employing this conditions higher activities were estimated in the commercial preparates as indicated. The high sensitivity commends this standardized method to use in melanoma research.

The cadmium content of 402 species of wild mushrooms was determined by flameless atomic absorption spectroscopy. Altogether 1049 samples, grown mainly, in southern Germany, were tested. The cadmium content was between less than 0,1 and 120 mg/kg dry weight, equivalent to less than 0,01 and 10,8 mg/kg fresh weight. Samples with low cadmium content were predominant: 68% of the samples contained less than 2 mg/kg dry weight, 86.5% contained less than 5 mg/kg dry weight, equivalent to about less than 0,2 and less than 0,5 mg/kg fresh weight, respectively. The cadmium content was clearly species-dependent, and to a lesser extent genus-dependent. Samples containing more than 10 mg/kg dry weight occurred in 41 species, among these were 9 Tricholomataceae, 10 Agaricaceae, 11 Cortinariaceae, 3 Amanitas and 4 Russula species. Samples containing more than 50 mg/kg dry weight were found in Agaricus augustus, A perrarus, A. silvicola, A. macrosporus, A. maleolens, and Inocybe bongardii. In single fruiting-bodies the lowest cadmium content was found in the stem, whereas the highest content was found in the gills and tubes. Cadmium content of the gills was at most five times the amount present in the cup. In cadmium-rich mushrooms a marked concentration as compared with the cadmium content of the soil had occured.

The inactivation of stationary phase cells by ultraviolet light (UV) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined in eight wild strains of Rhodotorula, six of which are the sporidial yeast phase of Rhodosporidium, a basidiomycetous fungus. It has been found that (1) the UV-resistance of Rhodosporidium and Rhodotorula yeasts is higher and the MNNG-resistance lower than the resistance of Candida and Hansenula yeasts, (2) the shape of the survival curves is sigmoid in the case of UV and two-phase exponential in the case of MNNG, (3) the mutagen sensitivities but not the inactivation kinetics of the strains are different, (4) the UV- and MNNG-sensitivities for each of the strains are correlated, (5) the relatively high resistance to UV cannot be due to the carotenoid pigments of the cells, (6) mutations to UV-sensitivity can be induced with a high rate, (7) the sigmoidal character of the UV survival curves were reduced or transformed to an exponential shape by the UVS-mutations.

The diagnosis of mushroom poisoning is based on three principles: the description of the mushroom, the toxicological analysis of the mushroom and, most important, the mushroom syndrome. Mushroom poisoning can be classified according to the lag time between the meal and the onset of symptoms. In this paper we will discuss mushroom poisoning with short and intermediate lag time. With short lag time and a predominance of CNS signs and symptoms the diagnosis is either fly-agaric, pantherina or psilocybin syndrome. Parasympathomimetic signs and symptoms indicate the muscarine syndrome. If--with a lag period of up to four hours--vomiting and diarrhea are predominant, we will find a poisoning with one of the many mushrooms which lead to gastroenteritis. Gastroenteritis combined with hemolysis points to a paxillus syndrome.

Three new antibiotics, merulinic acids A, B, and C, have been isolated from fruiting bodies of Merulis tremellosus and Phlebia radiata. They are closely related derivatives of beta-resorcylic and salicylic acid, carrying monounsaturated C17-alkyl side chains in 6-position. The merulinic acids are mixtures of the delta8'-compounds with minor amounts of the corresponding delta10'-, dehydro, and dihydro derivatives, separable only by means of HPLC. delta8'-Merulinic acid A (I) was synthesized via a biomimetic cyclization of a linear precursor. The merulinic acids inhibit a variety of bacteria and are not active against fungi. RNA, DNA, and protein synthesis in Bacillus brevis and Ehrlich carcinoma ascites cells are inhibited shortly after the addition of the antibiotics. Almost complete hemolysis of human erythrocytes is caused by 25 microgram/ml of merulinic acid B and C or by 73 microgram/ml of merulinic acid A.

In the second part of this review of mushroom poisonings, the syndromes with intermediate and long lag-times are discussed. They include the coprinus-, phalloides-, gyromitrin- and the orellanus syndrome. The coprinus syndrome occurs whenever alcohol is consumed after a meal containing coprine. The lag-time varies according to the amount and time of alcohol intake. It is very similar to the disulfiram syndrome which is known from the adverse therapy of alcoholism. The lag-time of the phalloides syndrome varies between 7 and 24 hours. It starts with massive gastroenteritis followed by hepatopathia which can lead to hepatic coma and kidney failure. The phalloides syndrome is caused by the amatoxins of the death caps, which inhibit the RNA Polymerase B in the nucleus of the liver cell. The gyromitrin syndrome exhibits also a delayed onset. The hepatotoxicity and the nephrotoxicity are less severe than in the phalloides syndrome. The first metabolite of gyromitrin monomethylhydrazin is responsible for CNS-symptoms such as delirium and convulsions. In contrast to the phalloides syndrome vomiting can be the only leading symptom in gyromitrin poisoning. The orellanus syndrome has the most delayed onset of all mushroom poisonings with 1-3 weeks. It should be thought of in all cases of kidney insufficiency of unknown origin. The orellanines damage the kidney and induce all degrees of kidney insufficiency according to the amount of ingested poison. Terminal kidney failure which requires hemodialysis treatment can occur in severe cases.

A Peroxidase (EC 1.11.1.7) of the basidiomycet Phellinus igniarius was derived from mycel and a medium containing glucose and extract of yeast by using various methods of preparation. The enzyme resists extreme conditions (pH, temperature salt concentration). Its optimum pH for activities is in the acid range. Two isoenzymes were found. The molecular weight, isoelectric point, Michaelis-Menten constant, indolacetic acid oxidase activity and spectral and analytical properties of this peroxidase were determined. It is assumed that the enzyme has an intracellular as well as an extracellular field of activity.

The lead content of 222 species of wild mushrooms, either edible or poisonous, was determined by graphite furnace atomic absorption spectroscopy. Altogether 494 samples, grown in rural regions of southern Germany, were tested. The lead content was between less than 2.0 and 40.4, on the average 10.6 mg/kg dry weight, equivalent to less than 0.1 and 3.0, on the average 1.06 mg/kg fresh weight.

Since 1982 we have performed surgery in 2 cases of obstruction ileus caused by fungi. Both patients had stenosis of the gut, due to adhesions and Crohn's disease respectively. Unless carefully chewed, mushrooms keep their shape and almost their size in the bowel, and can thus become jammed in a stenosis and cause ileus. High risk patients should be advised to avoid eating mushrooms or else to chew them very carefully.

A case is described of obstruction of the small bowel after consumption of 500 g edible mushrooms (Cantharellus cibarius). No fibrous bands or adhesions were found on laparotomy. When preparing the meal the mushrooms were not cut up, nor were they properly chewed due to the patient's defective dental prosthesis. 11 days after the meal the patient passed largely intact mushrooms. In retrospect the impacted mushrooms could be assumed on X-ray after barium meal. Since even without intra-abdominal adhesions impacted mushrooms can cause mechanical obstruction, it is recommended that mushrooms be cut up, chewed well and consumed only in moderate portions even when large quantities are available.

To carry out long-term experiments as part of a therapy concept of malignant tumours using inactive transport forms of cancerostatic substances and their specific cleavage in the acidic pH region of the tumours by application of extraneous enzymes, we require enzymes with similar catalytic and pharmacokinetic properties which differ from each other in immunological respect. In the search for such enzymes, the alpha-L-arabinofuranosidases from 12 different fungi, among them 9 basidiomycetes, were studied. The enzymes mentioned were demonstrable in all fungi. Optimum pH values ranged between 2.5 and 5.5. The Km values for the cleavage of alpha-L-arabinofuranoside were, in most cases, 0.5 to 1.8 moles-liter-1-10(-3). With regard to pH dependence, the alpha-L-arabinofuranosidases of most of the fungi investigated proved adequate for the long-term trials envisaged. 4-nitrophenyl-beta-D-glucopyranoside and -beta-cellobioside were also cleaved by enzyme preparations of all the 11 fungi investigated. The beta-D-glucopyranosidases showed a less favourable pH dependence than the alpha-L-arabinofuranosidases. The cleavage of 4-nitrophenyl-beta-cellobioside, on the contrary, showed mostly a comparatively favourable pH dependence. On the basis of the coinciding optimal pH values and the occurrence of 4-nitrophenyl-beta-D-glucopyranoside as an intermediate product in the cleavage of the corresponding cellobioside, we assume that both substrates are cleaved by beta-glucosidase. Because the occurrence of the glucoside during the cleavage of cellobioside is undesirable for the therapeutic trial, a method is proposed for selection of an appropriate cellobioside splitting enzyme basing on the present studies and the relevant literature.

According to recommended international standards edible fungi are blanched before salting and freezing. A study was conducted on the solution losses of Lactarius rufus due to blanching. Weight losses, changes of dry matter, raw fat, total nitrogen, amino nitrogen and ash contents as well as the pH value were determined when various methods of blanching were used. 3 min blanching at 95-100 degrees C was able to inactivate catalase and peroxydase while 6 min blanching was needed for inactivating polyphenoloxydase totally. After blanching there were 1/10 - 1/100 of spores left. During the 3 min blanching in water five times the quantity of mushrooms the losses of dry matter were about 10%; when doubling the quantity of blanching water the losses increased to 2-3 fold. The doubling of blanching time had no significant influence on the losses. The soluble dry matter content of blanched mushrooms was less than 50% of that of the fresh. Total nitrogen of fresh mushrooms was equal to that of the blanched but the amino nitrogen decreased to one tenth by blanching. The mineral element content of blanched mushrooms was about the half of that of the fresh. Blanching caused a slight decrease in the pH value. The necessity of the blanching of all edible fungi before freezing was discussed.

A procedure is described for the determination of agaritine in the commercial mushroom Agaricus bisporus by high performance liquid chromatography (HPLC). Agaritine was extracted from the mushroom sample with methanol and the filtered extract diluted with phosphate buffer. An aliquot of this solution was used directly for the HPLC-separation on a cation exchange column (Partisil SCX) with 0.5 mM phosphate buffer (pH 1.8) as mobile phase and u.v. monitoring at 237 nm. The agaritine content in fresh mushrooms was found to be in the range of 94-629 mg/kg fresh weight. Canned mushrooms contained 1-55 mg/kg drained weight with 3-103 mg/l in the liquid. The highest agaritine values were found in dried commercial mushrooms amounting to 2,110-6,905 mg/kg.

The lead-contents of canned and fresh mushrooms were analyzed by photometric determination after dry ashing and dithizone extraction. The average lead-content was found to be 0.49 ppm with extreme values of 0.10--1.0 ppm on fresh-weight basis resp. 5.5 ppm (2.0--13.5 ppm) on dry-weight basis. It appears that lead content increases with the size (age) of the mushrooms; the influence of canning upon the lead content is not yet finally clear, it seems, however, as if canning process has a lesser influence on the lead content than the size (age) of mushrooms.

A new edible mushroom, known as oyster mushroom (Pleutrotus Florida, variety of Pleurotus ostreatus) has recently gained commercial importance in Europe, having been imported from the U.S.A. in 1963 and cultivated in Germany between 1972 and 1973. Several persons concerned with the cultivation and industrial production of this mushroom fell ill, after close contact with it over a period of some months, with exhaustion, headache, chills and fever, and cough. These symptoms, similar to those of "farmer's lung" were proved to be caused by the spores of Pleurotus Florida, inhaled in harvesting rooms. The suspicion of an allergic disease, is supported by the history and clinical picture, and the demonstration of precipitating antibodies against spore extracts (Ouchterlony test). The antigens probably have a high molecular weight and are fixed to the spore membrane. Experimental sensitisation of rabbits also revealed precipitins. One of three isolated antigens produced antibodies identical in man and rabbit.

Primary hemolysis induced by antigens and toxins of mushrooms must be distinguished from hemolysis secondary to shock and disseminated intravascular coagulation with disruption of erythrocytes caused by severe poisoning with many mushroom species. Primary hemolysis is well documented as immunohemolysis after repeated ingestion of involute paxillus (Paxillus involutus). Direct hemolysis is reported after eating raw mushrooms with a high content of hemolysins. Hemolysis is only speculative in monomethylhydrazine poisoning by false morels (Gyromitra esculenta). Secondary hemolysis due to shock is not uncommon. Hemolysis in connection with enzymopenia of erythrocytes has not been documented as yet. In the present study the various hemolytic syndromes are described and discussed.