Fusarin C acts like an estrogenic agonist and stimulates breast cancer cells in vitro.

Fusarin C is a mycotoxin produced by several Fusarium species and has been associated with esophageal cancer due to its carcinogenic effects. Here, we report that fusarin C stimulates growth of the breast cancer cell line MCF-7. This suggests that fusarin C can act as an estrogenic agonist and should be classified as a mycoestrogen. MCF-7 cells were stimulated in the range between 0.1 and 20µM and inhibited when the concentration exceeded 50µM. The toxicity of fusarin C is comparable to other mycoestrogens such as zearalenone, but the chemical structure of fusarin C is very different from other known estrogen agonists. Furthermore, the toxicity of fusarin C was tested in five additional human cell lines Caco 2, U266, PC3, MDA-MB-231 and MCF-10a which were all inhibited when the concentration of fusarin C exceeded 10µM. To the best of our knowledge this is the first report on the mycoestrogenic properties of fusarin C.

Chromosome complement of the fungal plant pathogen Fusarium graminearum based on genetic and physical mapping and cytological observations.

A genetic map of the filamentous fungus Fusarium graminearum (teleomorph: Gibberella zeae) was constructed to both validate and augment the draft whole-genome sequence assembly of strain PH-1. A mapping population was created from a cross between mutants of the sequenced strain (PH-1, NRRL 31084, originally isolated from Michigan) and a field strain from Minnesota (00-676, NRRL 34097). A total of 111 ascospore progeny were analyzed for segregation at 235 loci. Genetic markers consisted of sequence-tagged sites, primarily detected as dCAPS or CAPS (n = 131) and VNTRs (n = 31), in addition to AFLPs (n = 66) and 7 other markers. While most markers exhibited Mendelian inheritance, segregation distortion was observed for 25 predominantly clustered markers. A linkage map was generated using the Kosambi mapping function, using a LOD threshold value of 3.5. Nine linkage groups were detected, covering 1234 cM and anchoring 99.83% of the draft sequence assembly. The nine linkage groups and the 22 anchored scaffolds from the sequence assembly could be assembled into four chromosomes, leaving only five smaller scaffolds (59,630 bp total) of the nuclear DNA unanchored. A chromosome number of four was confirmed by cytological karyotyping. Further analysis of the genetic map data identified variation in recombination rate in different genomic regions that often spanned several hundred kilobases.

Analysis of the structure and inheritance of a linear plasmid from the obligate biotrophic fungus Blumeria graminis f. sp. hordei.

A linear plasmid is widespread among isolates of the obligate biotrophic fungus Blumeria graminis f.sp. hordei (synonym Erysiphe graminis) (Bgh), the organism that causes the disease powdery mildew on barley. We cloned and sequenced the entire plasmid of 7965 bp. The plasmid contains two identical terminal inverted repeats (TIR) of 610 bp. Two ORFs are present on opposite strands, one encoding a phage-type DNA polymerase and the other a phage-type RNA polymerase. Two large transcripts of approximately 4.2 and 5.6 kb were identified in conidia, germinating conidia and Bgh -infected barley leaves, indicating that the polymerases are transcribed at most stages of the lifecycle. The transcription start sites were localised within the TIR regions, where a putative 11-bp ARS consensus sequence was also identified. To follow the sexual transmission of the plasmid we screened 27 Bgh isolates for mitochondrial polymorphisms. One polymorphism allowed us to carry out a cross between two isolates that differed in both mitochondrial genotype and presence/absence of the Bgh plasmid. The plasmid was transmitted independently of the origin of the mitochondria. No transfer of the plasmid was observed between two Bgh isolates that were co-cultivated for 1.5 years on a common susceptible barley variety. The plasmid appears to be an autonomous replicon with no phenotypic effect on Bgh.

*NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier.

To investigate the relevance of *NO and oxyradicals in the blood-brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (gamma-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced gamma-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn-, Cu/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 microM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in *NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature.

Astrocytes enhance radical defence in capillary endothelial cells constituting the blood-brain barrier.

Astrocytes (AC) induce blood-brain barrier (BBB) properties in brain endothelial cells (EC). As antioxidative activity (AOA) is assumed to be a BBB characteristic, we tested whether AC improve AOA of EC. Monocultivated AC showed higher AOA [manganese superoxide dismutase (SOD), catalase (Cat), glutathione peroxidase (GPx)] than EC. Cocultivation elevated AOA in EC (MnSOD, CuZnSOD, Cat, GPx), and AC (MnSOD, CuZnSOD, GPx). Hypoxia increased radical-induced membrane lipid peroxidation in monocultivated, but not in cocultivated EC. Thus, EC/AC cocultivation intensifies AOA in both cell types, protects the EC, and therefore, the BBB against oxidative stress. The high AOA is regarded as an essential property of the BBB, which is induced by AC.

Accelerated accumulation of somatic mutations in mice deficient in the nucleotide excision repair gene XPA.

Inheritable mutations in nucleotide excision repair (NER) genes cause cancer-prone human disorders, such as xeroderma pigmentosum, which are also characterized by symptoms of accelerated ageing. To study the impact of NER deficiency on mutation accumulation in vivo, mutant frequencies have been determined in liver and brain of 2-16 month old NER deficient XPA-/-, lacZ hybrid mice. While mutant frequencies in liver of 2-month old XPA-/-, lacZ mice were comparable to XPA+/-, lacZ and the lacZ parental strain animals, by 4 months of age mutant frequencies in the XPA-deficient mice were significantly increased by a factor of two and increased further until the age of 16 months. In brain, mutant frequencies were not found to increase with age. These results show that a deficiency in the NER gene XPA causes an accelerated accumulation of somatic mutations in liver but not in brain. This is in keeping with a higher incidence of spontaneous liver tumors reported earlier for XPA-/- mice after about 15 months of age.

Rapid accumulation of genome rearrangements in liver but not in brain of old mice.

Somatic mutations have long been considered a possible cause of ageing. To directly study mutational events in organs and tissues of ageing mammals, a transgenic mouse model has been generated that harbours lacZ reporter genes as part of chromosomally integrated plasmids. Using this model, we determined spontaneous mutant frequencies and spectra in mouse liver and brain as a function of age. In the liver, mutant frequencies increased with age from birth to 34 months; in the brain, an increase was observed only between birth and 4-6 months. Molecular characterization of the mutations showed that a substantial portion involved genome rearrangement events, with one breakpoint in a reporter gene and the other in the mouse flanking sequence. In the liver, these genome rearrangements did not increase with age until after 27 months, when they increased rapidly. In brain, the frequency of genome rearrangements was lower than in liver and did not increase with age.

Disparate sequence characteristics of the Erysiphe graminis f.sp. hordei glyceraldehyde-3-phosphate dehydrogenase gene.

The Erysiphe graminis f.sp. hordei (Egh) glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was isolated and characterized. It contains typical promoter elements and has three introns, one of which is positioned in the 5' untranslated region of the gene. The deduced amino-acid sequence has 87% similarity to gpd genes from other Ascomycete fungi. This is at the same level as previously estimated among these fungi. Comparison at the DNA level reveal similarities of only around 70%, which is 10% lower than previously reported. In an evolutionary tree based on the sequences from 18 fungal gpd genes, Egh falls into the group of Ascomycetes located at a basal position. The regulatory region of the Egh gpd gene has no homology to corresponding sequences in other filamentous Ascomycetes. Codon usage was determined for the four characterized Egh genes (tub2, Egh7, Egh16 and gpd) and found to be similar for all four genes. The results of the codon-usage analysis suggest that Egh is more flexible than other fungi in the choice of nucleotides at the wobble position. Codon-usage preferences in Egh and barley genes indicate a level of difference which may be exploited to discriminate between fungal and plant genes in sequence mixtures. The Egh gpd promoter appears to be superior to that of the Egh beta-tubulin gene (tub2) for driving the E. coli beta-glucuronidase (GUS) gene in transformation experiments.

Isolation and characterization of two novel genes expressed in germinating conidia of the obligate biotroph Erysiphe graminis f.sp. hordei.

A cDNA library was constructed from germinating conidia of the obligate biotrophic fungus, Erysiphe graminis DC ex Mérat f.sp. hordei Em. Marchal (Egh). Subtractive hybridization and differential screening were carried out. Two cDNA clones, cEgh7 and cEgh16, which were highly expressed in germinating conidia, but not in ungerminated conidia, were selected for further characterization. The corresponding genomic sequences, gEgh7 and gEgh16, were isolated from a cosmid library and sequenced. The gEgh7 gene contains an open reading frame (ORF) that codes for a 249-amino-acid (aa), Pro-rich polypeptide with a repeated primary structure. Expression studies in planta indicated that gEgh7 may have a function in the development and maturation of conidia. The ORF of gEgh16 is interrupted by two introns of 91 and 119 bp. It encodes a 251-aa polypeptide of unknown function. This gene belongs to a multigene family and is expressed during all developmental stages of Egh in planta and may be associated with hyphal growth.

Quantitative trait loci for heading date and straw characters in barley.

Quantitative trait loci (QTLs) for heading date and straw characters were examined in 79 chromosome-doubled haploid lines derived from the F1 generation of a cross between a six-rowed winter barley and a two-rowed spring barley. A genetic map covering 1100 cM containing 85 markers, including isozyme, morphological, RFLP, and RAPD markers, was constructed. All traits examined had two QTLs with large effects on chromosome 2. In addition, a QTL for length of the top internode was found on chromosome 6. The QTL in the chromosome segment around locus v (two row/six row) on chromosome 2 may be caused by pleiotropic effects of this locus. The same QTLs for heading date and straw length were found in both 1989 and 1991. The results indicate that two QTLs on chromosome 2 affect a group of correlated traits.

Biolistic transformation of the obligate plant pathogenic fungus, Erysiphe graminis f.sp. hordei.

Particle gun acceleration appears to be a possible way to transform mycelium cells of obligate plant parasites growing on host surfaces. GUS expression was obtained in E. graminis f.sp. hordei cells after bombardment with the GUS gene under the control of the E. graminis f.sp. hordei β-tubulin promoter. Three heterologous promoters, onefrom Aspergillus nidulans and two from Cochliobolus heterostrophus, gave very low or no expression of GUS.

Effect of MK-801 and U83836E on a porcine brain capillary endothelial cell barrier during hypoxia.

The present study investigated the influence of MK-801 (N-methyl-D-aspartate receptor antagonist) and U83836E (antioxidative aminosteroid) on the permeability of sodium fluorescein through a cell barrier during hypoxia (2 h 95% N2/5% CO2). The barrier consisted of porcine brain capillary endothelial cells and of cerebral rat astrocytes cultivated on two sides of a filter. After hypoxia, the permeation of fluorescein was significantly increased (10.2 +/- 1.5 x 10(-3) cm/min, P < 0.001) compared to the normoxic control (2 h 95% O2/5% CO2, 1.8 +/- 0.6 x 10(-3) cm/min). The hypoxia-enhanced permeation was significantly (P < 0.05) reduced by 10 microM MK-801 (2.0 +/- 0.5 x 10(-3) cm/min) and 10 microM U83836E (3.1 +/- 1.3 x 10(-3) cm/min). The results demonstrate, for the first time in a cell culture system, that hypoxia impairs brain endothelial barrier function, and that this enhanced permeability can be influenced pharmacologically. It is concluded that two distinct pathogenic mechanisms are involved in hypoxic cerebral endothelial cell injury, and that cerebroprotection afforded by these agents may result, in part, from reductions in edema secondary to improved blood-brain barrier function.

Genome manipulation in recalcitrant species: construction and characterization of a yeast artificial chromosome (YAC) library from Erysiphe graminis f. sp. hordei, an obligate fungal pathogen of barley.

Extraction of DNA from organisms where spores are the only source of pure material is a major problem. Methods are described which allow the isolation of high-M(r) DNA, from small quantities of Erysiphe graminis f. sp. hordei (Egh) conidia, suitable for cloning in yeast artificial chromosomes (YACs). A YAC library of 1500 clones was constructed in the vectors, pYAC4 and pYACRC. The average size of YAC inserts is 220 kb and range from 70 to 500 kb, providing ten haploid genome equivalents. Multicopy RFLP markers and an Egh-specific repetitive SINE element were used to characterize the library. The SINE element is effective in fingerprint analysis and contig assembly. Four out of five representative clones containing more than one YAC were mitotically unstable.

Distribution of RAPD markers on a linkage map of barley.

The RAPD technique was found to provide reliable genetic markers in barley. A linkage study of 23 RAPDs, 28 RFLPs, and 29 gene loci was conducted on 72 chromosome-doubled haploid progeny lines from a barley cross. The resulting linkage map covered 680 cM, about half of the barley genome. RAPD markers were distributed throughout the map, but a higher than expected frequency of tightly linked RAPDs was observed. Several cases of skewed segregation ratios were observed, but the RAPD markers segregated in ratios similar to their linked loci, confirming that they were reliably scored. In separate crosses, two amplified RAPD products, generated by different primers, were shown to reside in corresponding chromosomal positions. The RAPD markers seem a realistic alternative to RFLP markers in linkage analysis of barley.

Genetic diversity among wild and cultivated barley as revealed by RFLP.

Genetic variability of cultivated and wild barley, Hordeum vulgare ssp. vulgare and spontaneum, respectively, was assessed by RFLP analysis. The material consisted of 13 European varietes, single-plant offspring lines of eight land races from Ethiopia and Nepal, and five accessions of ssp. spontaneum from Israel, Iran and Turkey. Seventeen out of twenty-one studied cDNA and gDNA probes distributed across all seven barley chromosomes revealed polymorphism when DNA was digested with one of four restriction enzymes. A tree based on genetic distances using frequencies of RFLP banding patterns was estimated and the barley lines clustered into five groups reflecting geographical origin. The geographical groups of land-race lines showed less intragroup variation than the geographical groups of spontaneum lines. The group of European varieties, representing large variation in agronomic traits, showed an intermediate level. The proportion of gene diversity residing among geographical groups (FST) varied from 0.19 to 0.94 (average 0.54) per RFLP pattern, indicating large diversification between geographical groups.

SINE-like properties of a highly repetitive element in the genome of the obligate parasitic fungus Erysiphe graminis f.sp. hordei.

The genomic organization of repetitive DNA in the obligate parasitic fungus Erysiphe graminis DC ex Mérat f.sp. hordei Em. Marchal was investigated using a cosmid library of the fungal genome. Three repetitive sequences were shown to be dispersed throughout the genome, and in a few cases they were found closely associated with long poly(dA) tracts. The most prevalent sequence is 903 bp long and accounts for at least 5% of the genome. Sequence analysis revealed features resembling mammalian Short INterspersed Elements (SINEs), namely the presence of a poly(dA) tail (33 bp), flanking direct repeats (13 bp), putative "A" and "B" blocks for RNA polymerase III binding; the corresponding transcript would be capable of forming a complex secondary structure.

Localization of the Laevigatum powdery mildew resistance gene to barley chromosome 2 by the use of RFLP markers.

The powdery mildew disease resistance gene Ml(La) was found to belong to a locus on barely chromosome 2. We suggest that this locus be designated MlLa. Linkage analysis was carried out on 72 chromosome-doubled, spring-type progeny lines from a cross between the winter var 'Vogelsanger Gold' and the spring var 'Alf'. A map of chromosome 2 spanning 119cM and flanked by two peroxidase gene loci was constructed. In addition to the Laevigatum resistance locus the map includes nine RFLP markers, the two peroxidase gene loci and the six-row locus in barley.

Induction and purification of chitinase in Brassica napus L. ssp. oleifera infected with Phoma lingam.

A pathogen-induced chitinase (EC 3.2.1.14) was isolated from cotyledons of oilseed rape (Brassica napus cv. Bienvenu) 8 d after inoculation with Phoma lingam. The purified chitinase has a molecular weight of 30 kDa, and an isoelectric point of approx. 9.1. A partial amino-acid sequence obtained after tryptic digestion of the protein shows high sequence similarity to basic chitinases from bean, tobacco, potato, Arabidopsis, barley and rice, as well as to acidic chitinases from tobacco and petunia. A close serological relationship was found between the chitinase isoenzyme and an isoenzyme from sugar-beet (Beta vulgaris L.). When resistant and susceptible cultivars were inoculated with P. lingam there was a significant difference in the increase in chitinase activity during the early stage after inoculation. The resistant cultivars showed a rapid increase in chitinase activity, in contrast to susceptible cultivars where an increase in activity was delayed until 24 h after infection. By measuring the chitinase activity from the mycelium of P. lingam, it was concluded that the increase in chitinase activity found in infected plants was of plant origin. The chitinase activity was found to be restricted to the site of pathogen attack and was not systemically induced in other parts of the plant.