Damp building moulds: Assessment of sensitization in patients and studies into mechanisms of airway inflammation using experimental models

Exposure to water-damaged buildings and the associated health problems have evoked concern and created confusion during the past 20 years. Individuals exposed to moisture problem buildings report adverse health effects such as non-specific respiratory symptoms. Microbes, especially fungi, growing on the damp material have been considered as potential sources of the health problems encountered in these buildings. Fungi and their airborne fungal spores contain allergens and secondary metabolites which may trigger allergic as well as inflammatory types of responses in the eyes and airways. Although epidemiological studies have revealed an association between damp buildings and health problems, no direct cause-and-effect relationship has been established. Further knowledge is needed about the epidemiology and the mechanisms leading to the symptoms associated with exposure to fungi. Two different approaches have been used in this thesis in order to investigate the diverse health effects associated with exposure to moulds. In the first part, sensitization to moulds was evaluated and potential cross-reactivity studied in patients attending a hospital for suspected allergy. In the second part, one typical mould known to be found in water-damaged buildings and to produce toxic secondary metabolites was used to study the airway responses in an experimental model. Exposure studies were performed on both naive and allergen sensitized mice. The first part of the study showed that mould allergy is rare and highly dependent on the atopic status of the examined individual. The prevalence of sensitization was 2.7% to Cladosporium herbarum and 2.8% to Alternaria alternata in patients, the majority of whom were atopic subjects. Some of the patients sensitized to mould suffered from atopic eczema. Frequently the patients were observed to possess specific serum IgE antibodies to a yeast present in the normal skin flora, Pityrosporum ovale. In some of these patients, the IgE binding was partly found to be due to binding to shared glycoproteins in the mould and yeast allergen extracts. The second part of the study revealed that exposure to Stachybotrys chartarum spores induced an airway inflammation in the lungs of mice. The inflammation was characterized by an influx of inflammatory cells, mainly neutrophils and lymphocytes, into the lungs but with almost no differences in airway responses seen between the satratoxin producing and non-satratoxin producing strain. On the other hand, when mice were exposed to S. chartarum and sensitized/challenged with ovalbumin the extent of the inflammation was markedly enhanced. A synergistic increase in the numbers of inflammatory cells was seen in BAL and severe inflammation was observed in the histological lung sections. In conclusion, the results in this thesis imply that exposure to moulds in water damaged buildings may trigger health effects in susceptible individuals. The symptoms can rarely be explained by IgE mediated allergy to moulds. Other non-allergic mechanisms seem to be involved. Stachybotrys chartarum is one of the moulds potentially responsible for health problems. In this thesis, new reaction models for the airway inflammation induced by S. chartarum have been found using experimental approaches. The immunological status played an important role in the airway inflammation, enhancing the effects of mould exposure. The results imply that sensitized individuals may be more susceptible to exposure to moulds than non-sensitized individuals.

Microbiological, environmental and proteolytic aspects in chronic rhinosinusitis with nasal polyposis

Chronic rhinosinusitis is one of the most common chronic respiratory tract diseases affecting up to 15% of the adult population in the Western world. It may be perpetuated by factors predisposing to sinus ostial obstruction together with inflammatory changes in the sinus mucosa. Chronic rhinosinusitis is associated with asthma, and it may represent the same disease process. Chronic rhinosinusitis with nasal polyposis (CRSwNP) and asthma share also the characteristic inflammatory features and histopathologic feature of airway remodelling. Remodelling is considered as a key event in the pathogenesis of asthma. It is controlled by a delicate balance between the matrix metalloproteinases (MMPs) and their regulators. The purpose of the present study was to evaluate the microbiological findings, inflammatory features and MMP and tissue inhibitor of metalloproteinases-1 (TIMP-1) expression in CRSwNP. The results were related to the patient history, exposure to moisture and clinical outcome in order to find out possible explanations for the etiology and chronicity of CRSwNP. Bacterial culture results were similar in patients and in controls and do not explain the chronic course of CRSwNP. The presence of fungi seems to be more common in CRSwNP than chronic rhinosinusitis in general, and they should be actively searched for using microbiological as well as histological methods. Typical outdoor fungal species were found in nasal lavage samples taken from controls in the autumn but not in the winter, reflecting environmental exposure. Exposure to moisture was reported by 46% of the CRSwNP patients, which is in accordance to the Finnish general population. Exposed patients did not differ significantly from non-exposed subjects with regards to microbiological findings, tissue eosinophilia and clinical outcome. Significantly elevated levels of collagenase-2 (MMP-8) and interleukin (IL)-8 but not tumour necrosis factor-α were found in CRSwNP patients. In particular, the activation of mesenchymal-type MMP-8 but not polymorphonuclear-type MMP-8 was associated with elevated IL-8 levels. IL-8 and MMP-8 may form an inductive cytokine-proteinase cascade in CRSwNP pathogenesis and provide a target for novel therapies and a diagnostic tool for monitoring CRSwNP treatment. The proteolytic spectrum is different in eosinophilic and non-eosinophilic CRSwNP with the up-regulation of MMP-8 and MMP-9 in non-eosinophilic CRSwNP, suggesting different pathophysiology in these subgroups. The lack of MMP up-regulation was associated with a poor prognostic factor and worse clinical outcome, representing a possible synergic anti-inflammatory function of MMP-8 and MMP-9 in CRSwNP. This study provides new information about possible immunologic mechanisms in the pathogenesis of CRSwNP. The recently discovered anti-inflammatory/ defensive properties of MMP-8 and MMP-9 in animal models are reported for the first time in a clinical setting in human inflammatory diseases.

The key enzyme in galactose metabolism, UDP-galactose-4-epimerase, affects cell-wall integrity and morphology in Candida albicans even in the absence of galactose

The enzyme UDP-galactose-4-epimerase (GAL10) catalyzes a key step in galactose metabolism converting UDP-galactose to UDPglucose which then can get metabolized through glycolysis and TCA cycle thus allowing the cell to use galactose as a carbon and energy source. As in many fungi, a functional homolog of GAL10 exists in Candida albicans. The domainal organization of the homologs from Saccharomyces cerevisiae and C albicans show high degree of homology having both mutarotase and an epimerase domain. The former is responsible for the conversion of beta-D-galactose to alpha-D-galactose and the hitter for epimerization of UDP-galactose to UDP-glucose. Absence of C albicans GAL10 (CaGAL10) affects cell-wall organization, oxidative stress response, biofilm formation and filamentation. Cagal10 mutant cells tend to flocculate extensively as compared to the wild-type cells. The excessive filamentation in this mutant is reflected in its irregular and wrinkled colony morphology. Cagal10 strain is more susceptible to oxidative stress when tested in presence of H2O2. While the S. cerevsiae GAL10 (ScGAL10), essential for survival in the presence of galactose, has not been reported to have defects in the absence of galactose, the C albicans homolog shows these phenotypes during growth in the absence of galactose. Thus a functional CaGal10 is required not only for galactose metabolism but also for normal hyphal morphogenesis, colony morphology, maintenance of cell-wall integrity and for resistance to oxidative stress even in the absence of galactose. (c) 2006 Elsevier Inc. All rights reserved.

Characterization of hydrophobin proteins at interfaces and in solutions using x rays

Hydrophobins are a group of particularly surface active proteins. The surface activity is demonstrated in the ready adsorption of hydrophobins to hydrophobic/hydrophilic interfaces such as the air/water interface. Adsorbed hydrophobins self-assemble into ordered films, lower the surface tension of water, and stabilize air bubbles and foams. Hydrophobin proteins originate from filamentous fungi. In the fungi the adsorbed hydrophobin films enable the growth of fungal aerial structures, form protective coatings and mediate the attachment of fungi to solid surfaces. This thesis focuses on hydrophobins HFBI, HFBII, and HFBIII from a rot fungus Trichoderma reesei. The self-assembled hydrophobin films were studied both at the air/water interface and on a solid substrate. In particular, using grazing-incidence x-ray diffraction and reflectivity, it was possible to characterize the hydrophobin films directly at the air/water interface. The in situ experiments yielded information on the arrangement of the protein molecules in the films. All the T. reesei hydrophobins were shown to self-assemble into highly crystalline, hexagonally ordered rafts. The thicknesses of these two-dimensional protein crystals were below 30 Å. Similar films were also obtained on silicon substrates. The adsorption of the proteins is likely to be driven by the hydrophobic effect, but the self-assembly into ordered films involves also specific protein-protein interactions. The protein-protein interactions lead to differences in the arrangement of the molecules in the HFBI, HFBII, and HFBIII protein films, as seen in the grazing-incidence x-ray diffraction data. The protein-protein interactions were further probed in solution using small-angle x-ray scattering. Both HFBI and HFBII were shown to form mainly tetramers in aqueous solution. By modifying the solution conditions and thereby the interactions, it was shown that the association was due to the hydrophobic effect. The stable tetrameric assemblies could tolerate heating and changes in pH. The stability of the structure facilitates the persistence of these secreted proteins in the soil.

Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity

Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. These modifications are expected to affect not only the structure and physico-chemical properties of proteins but also their physiological characteristics, such as digestibility in the GI-tract and allergenicity. Protein cross-linking enzymes such as transglutaminases are currently commercially available, but also other types of cross-linking enzymes are being explored intensively. In this study, enzymatic cross-linking of β-casein, the most abundant bovine milk protein, was studied. Enzymatic cross-linking reactions were performed by fungal Trichoderma reesei tyrosinase (TrTyr) and the performance of the enzyme was compared to that of transglutaminase from Streptoverticillium mobaraense (Tgase). Enzymatic cross-linking reactions were followed by different analytical techniques, such as size exclusion chromatography -Ultra violet/Visible multi angle light scattering (SEC-UV/Vis-MALLS), phosphorus nuclear magnetic resonance spectroscopy (31P-NMR), atomic force (AFM) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). The research results showed that in both cases cross-linking of β-casein resulted in the formation of high molecular mass (MM ca. 1 350 kg mol-1), disk-shaped nanoparticles when the highest enzyme dosage and longest incubation times were used. According to SEC-UV/Vis-MALLS data, commercial β-casein was cross-linked almost completely when TrTyr and Tgase were used as cross-linking enzymes. In the case of TrTyr, high degree of cross-linking was confirmed by 31P-NMR where it was shown that 91 % of the tyrosine side-chains were involved in the cross-linking. The impact of enzymatic cross-linking of β-casein on in vitro digestibility by pepsin was followed by various analytical techniques. The research results demonstrated that enzymatically cross-linked β-casein was stable under the acidic conditions present in the stomach. Furthermore, it was found that cross-linked β-casein was more resistant to pepsin digestion when compared to that of non modified β-casein. The effects of enzymatic cross-linking of β-casein on allergenicity were also studied by different biochemical test methods. On the basis of the research results, enzymatic cross-linking decreased allergenicity of native β-casein by 14 % when cross-linked by TrTyr and by 6 % after treatment by Tgase. It can be concluded that in addition to the basic understanding of the reaction mechanism of TrTyr on protein matrix, the research results obtained in this study can have high impact on various applications like food, cosmetic, medical, textile and packing sectors.

The effects of abiotic and biotic factors on somatic embryogenesis and seedlings of Pinus sylvestris (L.)

Somatic embryogenesis (SE) is an asexual form of plant propagation that occurs in nature and mimics many of the events of sexual reproduction. Pinus sylvestris (L.) is an important source of timber in Northern Eurasia but it is recalcitrant to somatic embryogenesis. Several factors important for the success of the P. sylvestris embryogenic cultures have not been thoroughly investigated. In this study, we examined the effects of parental genotypes on the SE in P. sylvestris, the involvement of the gaseous plant growth regulator, ethylene in SE, and also biotic effects on somatic embryos as well as on seedlings. We tested parental effects on immature embryo initiation for different media, storage periods, and on the maturation process. Maternal effects were found to be crucial for SE in the absence of paternal effects. No maternal-paternal interaction was observed at any stage of somatic embryo production. Additionally the role of ethylene at different developmental stages of SE was investigated. Two ACC synthase genes, PsACS1 and PsACS2, were isolated and characterized. PsACS1 was expressed during the proliferation stage in all tested genotypes, whereas PsACS2 was only expressed in somatic embryos of each genotype. Ethylene production in embryos at stage 3 was significantly higher than the other stages. In a parallel study, the response of somatic embryos to fungal elicitors was investigated. Three fungi, a mutualistic ectomycorrhizal (ECM) fungus (Suillus bovinus), a weak Scots pine pathogen (Heterobasidion parviporum) and a strong pathogen (H. annosum) were used. The gene expression patterns for embryos exposed to the H. parviporum elicitor were found to be similar to that documented for S. bovinus among the tested genes. By contrast somatic embryos exposed to the H. annosum elicitor had a different pattern of regulation which was marked by a delayed response, and in some cases death of the embryos. Furthermore, interaction without direct contact between P. sylvestris seedlings and microbes (mutualistic and pathogenic fungus, cyanobacterium) were investigated. Several novel genes expressed in seedlings treated with ECM fungus were isolated which suggested that physical contact is not necessary for elicitation of host responses. The results suggest that somatic embryos and seedlings of P. sylvestris are genetically well equipped to respond to fungal elicitor/exudates and could serve as a suitable model for reproducible molecular studies in conifer tree patho- and symbiotic systems.

Wood-inhabiting Basidiomycetes in the Caucasus Region : Systematics and Biogeography

The Caucasus region is a hotspot of biodiversity and is one of the few areas in the Northern Hemisphere which harbor Pleistocene glacial refugia. The region encompasses Armenia, Azerbaijan, Georgia, the southernmost European Russia, NE Turkey, and northern Iran. The study on fungal composition of the Caucasus region and its connection and possible contribution to the present mycota of Europe has largely escaped empirical scrutiny. Using taxonomic surveys, phylogenetic reconstruction methods, haplotype analysis, and similarity tests, this study has aimed to, 1) summarize the knowledge on the occurrence of corticioids and polypores in the Caucasus region, 2) resolve the phylogenetic relationships of selected, resupinate wood-inhabiting basidiomycetes for which the Caucasus region is currently the mere, or one of the noteworthy areas of distribution, and, 3) assess the similarity of Caucasian corticioid fungi to those of Europe and important areas in the Northern Hemisphere, and to examine the significance of the Caucasus region as a glacial refugium for these fungi. This study provides the first catalogue of corticioids and polypores (635 species) occurring in the Caucasus region. The phylogeny and systematics of the Caucasian resupinate taxa in focus has been resolved and the usefulness of some morphological characters has been re-evaluated. In this context, four new genera and two new species were described and five new combinations were proposed, two of which were supplemented with modern descriptions. The species composition of corticioids in the Caucasus region is found to be distinctly more similar to Europe and North America than to East Asia and India. The highest molecular diversity and within population pairwise distance for Peniophorella praetermissa has been detected in the Caucasus and East Asia, with the isolates of the latter area being highly divergent from the European ones. This, and the assignment of root haplotype to the Caucasian isolates in a haplotype network for Phlebia tuberucalta and P. livida, call attention to the role of the Caucasus region in shaping the current mycota of Europe.

Trishanku, a novel regulator of cell-type stability and morphogenesis in Dictyostelium discoideum

We have identified a novel gene, trishanku (triA), by random insertional mutagenesis of Dictyostelium discoideum. TriA is a Broad complex Tramtrack bric-a-brac domain-containing protein that is expressed strongly during the late G2 phase of cell cycle and in presumptive spore (prespore (psp)) cells. Disrupting triA destabilizes cell fate and reduces aggregate size; the fruiting body has a thick stalk, a lowered spore: stalk ratio, a sub-terminal spore mass and small, rounded spores. These changes revert when the wild-type triA gene is re-expressed under a constitutive or a psp-specific promoter. By using short- and long-lived reporter proteins, we show that in triA(-) slugs the prestalk (pst)/psp proportion is normal, but that there is inappropriate transdifferentiation between the two cell types. During culmination, regardless of their current fate, all cells with a history of pst gene expression contribute to the stalk, which could account for the altered cell-type proportion in the mutant.

Discovery of oxidative enzymes for food engineering : Tyrosinase and sulfhydryl oxidase

Enzymes offer many advantages in industrial processes, such as high specificity, mild treatment conditions and low energy requirements. Therefore, the industry has exploited them in many sectors including food processing. Enzymes can modify food properties by acting on small molecules or on polymers such as carbohydrates or proteins. Crosslinking enzymes such as tyrosinases and sulfhydryl oxidases catalyse the formation of novel covalent bonds between specific residues in proteins and/or peptides, thus forming or modifying the protein network of food. In this study, novel secreted fungal proteins with sequence features typical of tyrosinases and sulfhydryl oxidases were iden-tified through a genome mining study. Representatives of both of these enzyme families were selected for heterologous produc-tion in the filamentous fungus Trichoderma reesei and biochemical characterisation. Firstly, a novel family of putative tyrosinases carrying a shorter sequence than the previously characterised tyrosinases was discovered. These proteins lacked the whole linker and C-terminal domain that possibly play a role in cofactor incorporation, folding or protein activity. One of these proteins, AoCO4 from Aspergillus oryzae, was produced in T. reesei with a production level of about 1.5 g/l. The enzyme AoCO4 was correctly folded and bound the copper cofactors with a type-3 copper centre. However, the enzyme had only a low level of activity with the phenolic substrates tested. Highest activity was obtained with 4-tert-butylcatechol. Since tyrosine was not a substrate for AoCO4, the enzyme was classified as catechol oxidase. Secondly, the genome analysis for secreted proteins with sequence features typical of flavin-dependent sulfhydryl oxidases pinpointed two previously uncharacterised proteins AoSOX1 and AoSOX2 from A. oryzae. These two novel sulfhydryl oxidases were produced in T. reesei with production levels of 70 and 180 mg/l, respectively, in shake flask cultivations. AoSOX1 and AoSOX2 were FAD-dependent enzymes with a dimeric tertiary structure and they both showed activity on small sulfhydryl compounds such as glutathione and dithiothreitol, and were drastically inhibited by zinc sulphate. AoSOX2 showed good stabil-ity to thermal and chemical denaturation, being superior to AoSOX1 in this respect. Thirdly, the suitability of AoSOX1 as a possible baking improver was elucidated. The effect of AoSOX1, alone and in combi-nation with the widely used improver ascorbic acid was tested on yeasted wheat dough, both fresh and frozen, and on fresh water-flour dough. In all cases, AoSOX1 had no effect on the fermentation properties of fresh yeasted dough. AoSOX1 nega-tively affected the fermentation properties of frozen doughs and accelerated the damaging effects of the frozen storage, i.e. giving a softer dough with poorer gas retention abilities than the control. In combination with ascorbic acid, AoSOX1 gave harder doughs. In accordance, rheological studies in yeast-free dough showed that the presence of only AoSOX1 resulted in weaker and more extensible dough whereas a dough with opposite properties was obtained if ascorbic acid was also used. Doughs containing ascorbic acid and increasing amounts of AoSOX1 were harder in a dose-dependent manner. Sulfhydryl oxidase AoSOX1 had an enhancing effect on the dough hardening mechanism of ascorbic acid. This was ascribed mainly to the produc-tion of hydrogen peroxide in the SOX reaction which is able to convert the ascorbic acid to the actual improver dehydroascorbic acid. In addition, AoSOX1 could possibly oxidise the free glutathione in the dough and thus prevent the loss of dough strength caused by the spontaneous reduction of the disulfide bonds constituting the dough protein network. Sulfhydryl oxidase AoSOX1 is therefore able to enhance the action of ascorbic acid in wheat dough and could potentially be applied in wheat dough baking.

Two Novel Hexadepsipeptides with Several Modified Amino Acid Residues Isolated from the Fungus

Two new cyclohexadepsipeptides have been isolated from the fungus Isaria. Fungal growth in solid media yielded hyphal strands from which peptide fractions were readily isolable by organic-solvent extraction. Two novel cyclodepsipeptides, isaridin A and isaridin B, have been isolated by reverse-phase HPLC, and characterized by ESI-MS and 1H-NMR. Single crystals of both peptides have been obtained, and their 3D structures were elucidated by X-ray diffraction. The isaridins contain several unusual amino acid residues. The sequences are cyclo(β-Gly-HyLeu-Pro-Phe-NMeVal-NMePhe) and cyclo(β-Gly-HyLeu-β-MePro-Phe-NMeVal-NMePhe), where NMeVal is N-methylvaline, NMePhe N-methylphenylalanine, and HyLeu hydroxyleucine (=2-hydroxy-4-methylpentanoic acid). The two peptides differ from one another at residue 3, isaridin A having an (S)-proline at this position, while β-methyl-(S)-proline (=(2S,3S)-2,3,4,5-tetrahydro-3-methyl-1H-pyrrole-2-carboxylic acid) is found in isaridin B. The solid-state conformations of both cyclic depsipeptides are characterized by the presence of two cis peptide bonds at HyLeu(2)-Pro(3)/HyLeu(2)-β-MePro(3) and NMeVal(5)-NMePhe(6), respectively. In isaridin A, a strong intramolecular H-bond is observed between Phe(4)CO⋅⋅⋅HNβ-Gly(1), and a similar, but weaker, interaction is observed between β-Gly(1)CO⋅⋅⋅HNPhe(4). In contrast, in isaridin B, only a single intramolecular H-bond is observed between β-Gly(1)CO⋅⋅⋅HNPhe(4

Studies on the macroconida of microsporum-canis-characteristics of invitro amino-acid incorporativing system

The characteristics of an in vitro polyuridylic acid dependent amino acid incorporating system prepared from germinating macroconidia of Microsporum canis are described. The incorporation of 14C-phenylalanine into polyphenylalanine is dependent on S-30 extract, adenosine triphosphate, magnesium ions and polyuridylic acid. Incorporation is slightly enhanced by yeast transfer ribonucleic acid and pyruvate kinase. The system is highly sensitive to ribonuclease, puromycin and miconazole (an antifungal agent), moderately sensitive to sodium fluoride and much less sensitive to phenethylalcohol, cycloheximide, chloramphenicol and deoxyribonuclease. Cell-free extract from ungerminated conidia has less capacity to synthesize the protein and during germination a marked increase in the protein synthetic activity is observed. The results from experiments wherein ribosomes and S-100 fraction from germinated and ungerminated spores are interchanged, revealed that the defect in the extract from the ungerminated spore is in the ribosomes.

The challenge of LC/MS/MS multi-mycotoxin analysis - Heracles battling the Hydra?

Mycotoxins are secondary metabolites of filamentous fungi. They pose a health risk to humans and animals due to their harmful biological properties and common occurrence in food and feed. Liquid chromatography/mass spectrometry (LC/MS) has gained popularity in the trace analysis of food contaminants. In this study, the applicability of the technique was evaluated in multi-residue methods of mycotoxins aiming at simultaneous detection of chemically diverse compounds. Methods were developed for rapid determination of toxins produced by fungal genera of Aspergillus, Fusarium, Penicillium and Claviceps from cheese, cereal based agar matrices and grains. Analytes were extracted from these matrices with organic solvents. Minimal sample clean-up was carried out before the analysis of the mycotoxins with reversed phase LC coupled to tandem MS (MS/MS). The methods were validated and applied for investigating mycotoxins in cheese and ergot alkaloid occurrence in Finnish grains. Additionally, the toxin production of two Fusarium species predominant in northern Europe was studied. Nine mycotoxins could be determined from cheese with the method developed. The limits of quantification (LOQ) allowed the quantification at concentrations varying from 0.6 to 5.0 µg/kg. The recoveries ranged between 96 and 143 %, and the within-day repeatability (as relative standard deviation, RSDr) between 2.3 and 12.1 %. Roquefortine C and mycophenolic acid could be detected at levels of 300 up to 12000 µg/kg in the mould cheese samples analysed. A total of 29 or 31 toxins could be analysed with the method developed for agar matrices and grains, with the LOQs ranging overall from 0.1 to 1250 µg/kg. The recoveries ranged generally between 44 and 139 %, and the RSDr between 2.0 and 38 %. Type-A trichothecenes and beauvericin were determined from the cereal based agar and grain cultures of F. sporotrichioides and F. langsethiae. T-2 toxin was the main metabolite, the average levels reaching 22000 µg/kg in the grain cultures after 28 days of incubation. The method developed for ten ergot alkaloids from grains allowed their quantification at levels varying from 0.01 to 10 µg/kg. The recoveries ranged from 51 to 139 %, and the RSDr from 0.6 to 13.9 %. Ergot alkaloids were measured in barley and rye at average levels of 59 and 720 µg/kg, respectively. The two most prevalent alkaloids were ergocornine and ergocristine. The LC/MS methods developed enabled rapid detection of mycotoxins in such applications where several toxins co-occurred. Generally, the performance of the methods was good, allowing reliable analysis of the mycotoxins of interest with sufficiently low quantification limits. However, the variation in validation results highlighted the challenges related to optimising this type of multi-residue methods. New data was obtained about the occurrence of mycotoxins in mould cheeses and of ergot alkaloids in Finnish grains. In addition, the study revealed the high mycotoxin-producing potential of two common fungi in Finnish crops. The information can be useful when risks related to fungal and mycotoxin contamination will be assessed.

Studies on the macroconidia of Microsporum canis

The characteristics of an in vitro polyuridylic acid dependent amino acid incorporating system prepared from germinating macroconidia of Microsporum canis are described. The incorporation of 14C-phenylalanine into polyphenylalanine is dependent on S-30 extract, adenosine triphosphate, magnesium ions and polyuridylic acid. Incorporation is slightly enhanced by yeast transfer ribonucleic acid and pyruvate kinase. The system is highly sensitive to ribonuclease, puromycin and miconazole (an antifungal agent), moderately sensitive to sodium fluoride and much less sensitive to phenethylalcohol, cycloheximide, chloramphenicol and deoxyribonuclease. Cell-free extract from ungerminated conidia has less capacity to synthesize the protein and during germination a marked increase in the protein synthetic activity is observed. The results from experiments wherein ribosomes and S-100 fraction from germinated and ungerminated spores are interchanged, revealed that the defect in the extract from the ungerminated spore is in the ribosomes.

A linear sequence of extant families and genera of lycophytes and ferns

Throughout the history of the classification of extant ferns (monilophytes) and lycophytes, familial and generic concepts have been in great flux. For the organisation of lycophytes and ferns in herbaria, books, checklists, indices and spore banks and on the internet, this poses a problem, and a standardized linear sequence of these plants is therefore in great need. We provide here a linear classification to the extant lycophytes and ferns based on current phylogenetic knowledge; this provides a standardized guide for organisation of fern collections into a more natural sequence. Two new families, Diplaziopsidaceae and Rhachidosoraceae, are here introduced.