Manganese and zinc toxicity thresholds for mountain and Geyer willow

Information on the heavy metal toxicity thresholds of woody species endemic to the western United States is lacking but critical for successful restoration of contaminated riparian areas. Manganese (Mn, 50-10,000 mg l(-1)) and zinc (Zn, 100-1000 mg l(-1)) toxicity thresholds were determined for Geyer (Salix geyeriana Anderss.) and mountain (S. monticola Bebb) willow using a sand-culture technique. The lethal concentration (50%) values were 3117 and 2791 mg Mn l(-1) and 556 and 623 mg Zn l(-1) for Geyer and mountain willow, respectively. The effective concentration (50%) values for shoots were 2263 and 1027 mg Mn l(-1) and 436 and 356 mg Zn l(-1) for Geyer and mountain willow, respectively. Shoot tissue values did not increase with increasing treatment concentrations. However, metals in the roots did increase consistently in response to the treatments. Metal levels in the shoot tissues were low for Zn (65-139 mg kg(-1)) and moderate for Mn (1300-2700 mg kg(-1)). Geyer and mountain willow have good resistance to Mn, possibly due to evolution in hydric soils with increased Mn availability, and may be useful for phytostabilization of soils with high levels of available Mn. Both species were affected to a greater degree by Zn as compared to Mn, but still exhibited good resistance and should be useful in remediating sites with at least moderate levels of available Zn. Based on the thresholds evaluated, Geyer willow had greater resistance to both Mn and Zn as compared to mountain willow, especially at lower concentrations in which growth of Geyer willow was actually stimulated.     

Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy

Submicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-l-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1- to 2-μm fragments, compared to 100% of >2-μm fragments generated from pure freeze-dried mycelial fragments of A. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample.     

Universal and confident phosphorylation site localization using phosphoRS

An algorithm for the assignment of phosphorylation sites in peptides is described. The program uses tandem mass spectrometry data in conjunction with the respective peptide sequences to calculate site probabilities for all potential phosphorylation sites. Tandem mass spectra from synthetic phosphopeptides were used for optimization of the scoring parameters employing all commonly used fragmentation techniques. Calculation of probabilities was adapted to the different fragmentation methods and to the maximum mass deviation of the analysis. The software includes a novel approach to peak extraction, required for matching experimental data to the theoretical values of all isoforms, by defining individual peak depths for the different regions of the tandem mass spectrum. Mixtures of synthetic phosphopeptides were used to validate the program by calculation of its false localization rate versus site probability cutoff characteristic. Notably, the empirical obtained precision was higher than indicated by the applied probability cutoff. In addition, the performance of the algorithm was compared to existing approaches to site localization such as Ascore. In order to assess the practical applicability of the algorithm to large data sets, phosphopeptides from a biological sample were analyzed, localizing more than 3000 nonredundant phosphorylation sites. Finally, the results obtained for the different fragmentation methods and localization tools were compared and discussed.     

Substrate-independent approach for the generation of functional protein resistant surfaces

A new route for coating various substrates with antifouling polymer layers was developed. It consisted in deposition of an amino-rich adhesion layer by means of RF magnetron sputtering of Nylon 6,6 followed by the well-controlled, surface-initiated atom transfer radical polymerization of antifouling polymer brushes initiated by bromoisobutyrate covalently attached to amino groups present in the adhesion layer. Polymer brushes of hydroxy- and methoxy-capped oligoethyleneglycol methacrylate and carboxybetaine acrylamide were grafted from bromoisobutyrate initiator attached to a 15 nm thick amino-rich adhesion layer deposited on gold, silicon, polypropylene, and titanium-aluminum-vanadium alloy surfaces. Well-controlled polymerization kinetics made it possible to control the thickness of the brushes at a nanometer scale. Zero fouling from single protein solutions and a reduction of more than 90% in the fouling from blood plasma observed on the uncoated surfaces was achieved. The feasibility of functionalization with bioactive compounds was tested by covalent attachment of streptavidin onto poly(oligoethylene glycol methacrylate) brush and subsequent immobilization of model antibodies and oligonucleotides. The procedure is nondestructive and does not require any chemical preactivation or the presence of reactive groups on the substrate surface. Contrary to current antifouling modifications, the developed coating can be built on various classes of substrates and preserves its antifouling properties even in undiluted blood plasma. The new technique might be used for fabrication of biotechnological and biomedical devices with tailor-made functions that will not be impaired by fouling from ambient biological media.     

A novel mutation in the mitochondrial tRNAAla gene (m.5636T>C) in a patient with progressive external ophthalmoplegia

We report a heteroplasmic novel mutation m.5636T>C in the mt-tRNA^Ala in a patient with bilateral ptosis and ophthalmoparesis in whom a muscle biopsy showed cytochrome c oxdidase (COX) negative and ragged red fibers. Using laser capture microdissection we have isolated COX negative fibers and COX positive fibers from the muscle of the patient and determined that the mutation load was clearly increased in COX negative muscle fibers. Additionally, the mutated m.5636T nucleotide is conserved in all the mammal and non-mammal species analyzed and might be structurally relevant as it is located in a position involved in the formation of tertiary structure of canonical mitochondrial tRNAs.     

The targeting of plasmalemmal ceramide to mitochondria during apoptosis

Ceramide is a key lipid mediator of cellular processes such as differentiation, proliferation, growth arrest and apoptosis. During apoptosis, ceramide is produced within the plasma membrane. Although recent data suggest that the generation of intracellular ceramide increases mitochondrial permeability, the source of mitochondrial ceramide remains unknown. Here, we determine whether a stress-mediated plasmalemmal pool of ceramide might become available to the mitochondria of apoptotic cells. We have previously established annexin A1--a member of a family of Ca(2+) and membrane-binding proteins--to be a marker of ceramide platforms. Using fluorescently tagged annexin A1, we show that, upon its generation within the plasma membrane, ceramide self-associates into platforms that subsequently invaginate and fuse with mitochondria. An accumulation of ceramide within the mitochondria of apoptotic cells was also confirmed using a ceramide-specific antibody. Electron microscopic tomography confirmed that upon the formation of ceramide platforms, the invaginated regions of the plasma membrane extend deep into the cytoplasm forming direct physical contacts with mitochondrial outer membranes. Ceramide might thus be directly transferred from the plasma membrane to the mitochondrial outer membrane. It is conceivable that this "kiss-of-death" increases the permeability of the mitochondrial outer membrane thereby triggering apoptosis.     

Increased power of microarray analysis by use of an algorithm based on a multivariate procedure

MOTIVATION: The power of microarray analyses to detect differential gene expression strongly depends on the statistical and bioinformatical approaches used for data analysis. Moreover, the simultaneous testing of tens of thousands of genes for differential expression raises the 'multiple testing problem', increasing the probability of obtaining false positive test results. To achieve more reliable results, it is, therefore, necessary to apply adjustment procedures to restrict the family-wise type I error rate (FWE) or the false discovery rate. However, for the biologist the statistical power of such procedures often remains abstract, unless validated by an alternative experimental approach. RESULTS: In the present study, we discuss a multiplicity adjustment procedure applied to classical univariate as well as to recently proposed multivariate gene-expression scores. All procedures strictly control the FWE. We demonstrate that the use of multivariate scores leads to a more efficient identification of differentially expressed genes than the widely used MAS5 approach provided by the Affymetrix software tools (Affymetrix Microarray Suite 5 or GeneChip Operating Software). The practical importance of this finding is successfully validated using real time quantitative PCR and data from spike-in experiments. AVAILABILITY: The R-code of the statistical routines can be obtained from the corresponding author. CONTACT: Schuster@imise.uni-leipzig.de