A survey on the occurrence of mycotoxins in wheat and maize from western Romania

Samples of wheat (n = 25) and maize (n = 30) for animal consumption, collected in 1997 after harvest from western Romania, were analyzed by enzyme immunoassays for mycotoxin contamination. Toxins analyses included deoxynivalenol (DON), 3-acetylDON, 15- acetylDON, fusarenone X (FX), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), zearalenone (ZEA), fumonisin B1 (FB1), aflatoxin B1 (AFB1), ochratoxin A (OA), and citrinin (CT). DON and acetylDONs were the major contaminants in wheat (100%) and maize (46%). Median values for DON, 3-acetylDON, and 15-acetylDON were 880 μg kg-1, 66 μg kg- 1, and 150 μg kg-1 in wheat, and 890 μg kg-1, 180 μg kg-1, and 620 μg kg- 1 in maize, respectively. Additionally, 3,15-diacetylDON was detected in some samples by HPLC-EIA analysis. All samples were negative for FX (<150 μg kg-1). T-2 was found in wheat (n = 6) and maize (n = 1) at levels between 13 and 63 μg kg- 1. DAS (2.6 μg kg-1) was found in one maize sample. ZEA occurred in all wheat and in four maize samples, median values were 10 μg kg-1 and 250 μg kg-1, respectively. One maize sample contained FB1 (140 μg kg-1). All samples were AFB1-negative (<4 μg kg-1). OA was found in one wheat sample (37 μg kg- 1), CT was found in one maize sample (580 μg kg- 1). This first reported natural occurrence of a range of mycotoxins in Romanian feeding stuff shows that DON and acetyl DONs may be present at levels which may affect animal production. This revised version was published online in June 2006 with corrections to the Cover Date.     

On-line melting of double-stranded DNA for analysis of single-stranded DNA using capillary electrophoresis

Capillary electrophoresis (CE) is a convenient, fast and non-radioactive method with possibilities for automatization. To analyse single-stranded DNA molecules in a more automated way, we developed a heating device to melt double-stranded DNA fragments in the capillary during electrophoresis. In this study we used this device to obtain single-stranded DNA, necessary for the detection of point mutations in DNA using the single-strand conformation polymorphism technique. Results show that double-stranded DNA molecules can be melted on-line into single-stranded DNA molecules, although not for 100%. In an attempt to find universal electrophoretic conditions for the analysis of single-stranded DNA, we investigated the influence of several parameters on the yield of single-stranded DNA molecules and on the resolution of the single-stranded DNA peaks. We demonstrate that this heating device is a technical adjustment of CE which contributes to more automated analyses of DNA fragments.     

Specific proteins in the sieve-tube exudate of Ricinus commuais L. seedlings: separation,characterization and in-vivo labelling

Ricinus communis L. seedlings exuded pure phloem sap from the cut hypocotyl for several hours. Throughout the entire exudation period proteins were present in the phloem exudate at a constant concentration ranging from 0.11 to 0.41 mg·ml^–1 depending on the culture conditions and the age of the seedlings. Manipulation of the nutrient supply at the cotyledons after removal of the endosperm did not change the protein concentration in the exudate. Comparison of sieve-tube exudate proteins (STEPs) with soluble proteins extracted from the hypocotyl and the cotyledons showed a unique abundance of small proteins in the exudate, with molecular weights ranging from 10 to 25 kDa. Bands at 18, 19 and 20 kDa were especially dominant. The proteins found transiently in the xylem exudate, which might represent proteins secreted at the wound surface, were different in pattern. Two-dimensional separation of STEPs revealed that more than 100 distinct polypeptides occurred in the sieve-tube exudate, most of them slightly acidic with isoelectric points ranging from 4 to 6 and a few basic ones around 8. [³⁵S]Methionine fed to the cotyledons led to labelling of STEPs, demonstrating their rapid synthesis. It is concluded that there is a continuous synthesis and translocation of specific sieve-tube proteins, whose function is unknown.Abbreviations IEF isoelectric focussing - pI isoelectric point - STEP sieve-tube exudate protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid We wish to thank Pia Großmann and Libuse Badewitz for technical help.     

Detection of Indigenous Halobacillus Populations in Damaged Ancient Wall Paintings and Building Materials: Molecular Monitoring and Cultivation

Several moderately halophilic gram-positive, spore-forming bacteria have been isolated by conventional enrichment cultures from damaged medieval wall paintings and building materials. Enrichment and isolation were monitored by denaturing gradient gel electrophoresis and fluorescent in situ hybridization. 16S ribosomal DNA analysis showed that the bacteria are most closely related to Halobacillus litoralis. DNA-DNA reassociation experiments identified the isolates as a population of hitherto unknown Halobacillus species.     

Diurnal changes in assimilate concentrations and fluxes in the phloem of castor bean (Ricinus communis L.) and tansy (Tanacetum vulgare L.)

Reports about diurnal changes of assimilates in phloem sap are controversial. We determined the diurnal changes of sucrose and amino acid concentrations and fluxes in exudates from cut aphid stylets on tansy leaves (Tanacetum vulgare), and sucrose, amino acid and K(+) concentrations and fluxes in bleeding sap of castor bean pedicel (Ricinus communis). Approximately half of the tansy sieve tubes exhibited a diurnal cycle of sucrose concentrations and fluxes in phloem sap. Data from many tansy plants indicated an increased sucrose flux in the phloem during daytime in case of low N-nutrition, not at high N-nutrition. The sucrose concentration in phloem sap of young Ricinus plants changed marginally between day and night, whereas the sucrose flux increased 1.5-fold during daytime (but not in old Ricinus plants). The amino acid concentrations and fluxes in tansy sieve tubes exhibited a similar diurnal cycle as the sucrose concentrations and fluxes, including their dependence on N-nutrition. The amino acid fluxes, but not the concentrations, in phloem sap of Ricinus were higher at daytime. The sucrose/amino acid ratio showed no diurnal cycle neither in tansy nor in Ricinus. The K(+)-concentrations in phloem sap of Ricinus, but not the K(+) fluxes, decreased slightly during daytime and the sucrose/K(+)-ratio increased. In conclusion, a diurnal cycle was observed in sucrose, amino acid and K(+) fluxes, but not necessarily in concentrations of these assimilates. Because of the large variations between different sieve tubes and different plants, the nutrient delivery to sink tissues is not homeostatic over time.     

Geographical adaptation prevails over species‐specific determinism in trees’ vulnerability to climate change at Mediterranean rear‐edge forests

Climate change may reduce forest growth and increase forest mortality, which is connected to high carbon costs through reductions in gross primary production and net ecosystem exchange. Yet, the spatiotemporal patterns of vulnerability to both short‐term extreme events and gradual environmental changes are quite uncertain across the species’ limits of tolerance to dryness. Such information is fundamental for defining ecologically relevant upper limits of species tolerance to drought and, hence, to predict the risk of increased forest mortality and shifts in species composition. We investigate here to what extent the impact of short‐ and long‐term environmental changes determines vulnerability to climate change of three evergreen conifers (Scots pine, silver fir, Norway spruce) and two deciduous hardwoods (European beech, sessile oak) tree species at their southernmost limits of distribution in the Mediterranean Basin. Finally, we simulated future forest growth under RCP 2.6 and 8.5 emission scenarios using a multispecies generalized linear mixed model. Our analysis provides four key insights into the patterns of species’ vulnerability to climate change. First, site climatic marginality was significantly linked to the growth trends: increasing growth was related to less climatically limited sites. Second, estimated species‐specific vulnerability did not match their a priori rank in drought tolerance: Scots pine and beech seem to be the most vulnerable species among those studied despite their contrasting physiologies. Third, adaptation to site conditions prevails over species‐specific determinism in forest response to climate change. And fourth, regional differences in forests vulnerability to climate change across the Mediterranean Basin are linked to the influence of summer atmospheric circulation patterns, which are not correctly represented in global climate models. Thus, projections of forest performance should reconsider the traditional classification of tree species in functional types and critically evaluate the fine‐scale limitations of the climate data generated by global climate models.     

The role of c-FLIP splice variants in urothelial tumours

Deregulation of apoptosis is common in cancer and is often caused by overexpression of anti-apoptotic proteins in tumour cells. One important regulator of apoptosis is the cellular FLICE-inhibitory protein (c-FLIP), which is overexpressed, for example, in melanoma and Hodgkin''s lymphoma cells. Here, we addressed the question whether deregulated c-FLIP expression in urothelial carcinoma impinges on the ability of death ligands to induce apoptosis. In particular, we investigated the role of the c-FLIP splice variants c-FLIP_long (c-FLIP_L) and c-FLIP_short (c-FLIP_S), which can have opposing functions. We observed diminished expression of the c-FLIP_L isoform in urothelial carcinoma tissues as well as in established carcinoma cell lines compared with normal urothelial tissues and cells, whereas c-FLIP_S was unchanged. Overexpression and RNA interference studies in urothelial cell lines nevertheless demonstrated that c-FLIP remained a crucial factor conferring resistance towards induction of apoptosis by death ligands CD95L and TRAIL. Isoform-specific RNA interference showed c-FLIP_L to be of particular importance. Thus, urothelial carcinoma cells appear to fine-tune c-FLIP expression to a level sufficient for protection against activation of apoptosis by the extrinsic pathway. Therefore, targeting c-FLIP, and especially the c-FLIP_L isoform, may facilitate apoptosis-based therapies of bladder cancer in otherwise resistant tumours.     

Taxonomic and biogeographic patterns in the native and alien woody flora of Kashmir Himalaya,India

Biodiversity inventories unravel insightful taxonomic and biogeographic patterns that provide valuable inputs in guiding conservation practices and policymaking. Here we present an updated inventory of the native and alien woody flora of the Kashmir Himalaya, India, together with analyses of the patterns of taxonomic composition, geographic distribution, and invasion status of the alien species. The data was assembled from the floristic literature of the last two centuries, supplemented with herbarium records and validated by field surveys over the past decade. In total, the woody flora comprised of 520 species; out of these 322 species were native and 198 alien, yielding a proportion of 38% aliens. Amongst the natives, 16 species are on the IUCN red list 2009. While 37 genera included both native and alien species indicating a potential for hybrid formation, only 15 families were represented by alien species exclusively. Regarding the invasion status, presently 37 alien woody species are naturalized and 7 are invasive in the region. The results show that the Kashmir Himalaya harbours a diverse woody flora. It is concluded that a distinction between the native and the alien flora, as adopted in the present study, is crucial for a meaningful analysis of any biodiversity patterns. We predict that in the near future many alien woody species, presently naturalized in the region, can become serious plant invaders, and therefore need further research efforts and timely management.     

Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering

We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and quaternary conformational changes of human hemoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. We also report data on optically induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide applicability of the technique. By providing insights into the structural dynamics of proteins functioning in their natural environment, TR-WAXS complements and extends results obtained with time-resolved optical spectroscopy and X-ray crystallography.     

Functional characterization of Delta3,Delta2-enoyl-CoA isomerases from rat liver.

The degradation of unsaturated fatty acids by beta-oxidation involves Delta(3),Delta(2)-enoyl-CoA isomerases (enoyl-CoA isomerases) that catalyze 3-cis --> 2-trans and 3-trans --> 2-trans isomerizations of enoyl-CoAs and the 2,5 --> 3,5 isomerization of dienoyl-CoAs. An analysis of rat liver enoyl-CoA isomerases revealed the presence of a monofunctional enoyl-CoA isomerase (ECI) in addition to mitochondrial enoyl-CoA isomerase (MECI) in mitochondria, whereas peroxisomes contain ECI and multifunctional enzyme 1 (MFE1). Thus ECI, which previously had been described as peroxisomal enoyl-CoA isomerase, was found to be present in both peroxisomes and mitochondria. This enzyme seems to be identical with mitochondrial long-chain enoyl-CoA isomerase (Kilponen, J.M., Palosaari, P.M., and Hiltunen, J.K. 1990. Biochem. J. 269, 223-226). All three hepatic enoyl-CoA isomerases have broad chain length specificities but are distinguishable by their preferences for one of the three isomerization reactions. MECI is most active in catalyzing the 3-cis --> 2-trans isomerization; ECI has a preference for the 3-trans --> 2-trans isomerization, and MFE1 is the optimal isomerase for the 2,5 --> 3,5 isomerization. A functional characterization based on substrate specificities and total enoyl-CoA isomerase activities in rat liver leads to the conclusion that the 3-cis --> 2-trans and 2,5 --> 3,5 isomerizations in mitochondria are catalyzed overwhelmingly by MECI, whereas ECI contributes significantly to the 3-trans --> 2-trans isomerization. In peroxisomes, ECI is predicted to be the dominant enzyme for the 3-cis --> 2-trans and 3-trans --> 2-trans isomerizations of long-chain intermediates, whereas MFE1 is the key enzyme in the 2,5 --> 3,5 isomerization.