1,4-Naphthoquinones as inducers of oxidative damage and stress signaling in HaCaT human keratinocytes

Selected biological effects of 1,4-naphthoquinone, menadione (2-methyl-1,4-naphthoquinone) and structurally related quinones from natural sources - the 5-hydroxy-naphthoquinones juglone, plumbagin and the 2-hydroxy-naphthoquinones lawsone and lapachol - were studied in human keratinocytes (HaCaT). 1,4-naphthoquinone and menadione as well as juglone and plumbagin were highly cytotoxic, strongly induced reactive oxygen species (ROS) formation and depleted cellular glutathione. Moreover, they induced oxidative DNA base damage and accumulation of DNA strand breaks, as demonstrated in an alkaline DNA unwinding assay. Neither lawsone nor lapachol (up to 100 μM) were active in any of these assays. Cytotoxic and oxidative action was paralleled by stimulation of stress signaling: all tested quinones except lawsone and lapachol strongly induced phosphorylation of the epidermal growth factor receptor (EGFR) and the related ErbB2 receptor tyrosine kinase. EGFR activation by plumbagin, juglone and menadione was attenuated by a superoxide dismutase mimetic, indicating that ROS-related mechanisms contribute to EGFR activation by these naphthoquinones.     

Catalase evolved to concentrate H2O2 at its active site

Catalase is a homo-tetrameric enzyme that has its heme active site deeply buried inside the protein. Its only substrate, hydrogen peroxide (H₂O₂), reaches the heme through a 45 Å-long channel. Large-subunit catalases, but not small-subunit catalases, have a loop (gate loop) that interrupts the major channel. Two accesses lead to a gate that opens the final section of the channel to the heme; gates from the R-related subunits are interconnected. Using molecular dynamic simulations of the Neurospora crassa catalase-1 tetramer in a box of water (48,600 molecules) or 6 M H₂O₂, it is shown that the number of H₂O₂ molecules augments at the surface of the protein and in the accesses to the gate and the final section of the channel. Increase in H₂O₂ is due to the prevalence and distribution of amino acids that have an increased residency for H₂O₂ (mainly histidine, proline and charged residues), which are localized at the protein surface and the accesses to the gate. In the section of the channel from the heme to the gate, turnover rate of water molecules was faster than for H₂O₂ and increased residence sites for water and H₂O₂ were determined. In the presence of H₂O₂, the exclusion of water molecules from a specific site suggests a mechanism that could contend with the competing activity of water, allowing for catalase high kinetic efficiency.     

Integrin-linked kinase controls microtubule dynamics required for plasma membrane targeting of caveolae

Caveolae are specialized compartments of the plasma membrane that are involved in signaling, endocytosis, and cholesterol transport. Their formation requires the transport of caveolin-1 to the plasma membrane, but the molecular mechanisms regulating the transport are largely unknown. Here, we?identify a critical role for adhesion-mediated signaling through β1 integrins and integrin-linked kinase (ILK) in caveolae formation. Mice lacking β1 integrins or ILK in keratinocytes have dramatically reduced numbers of plasma membrane caveolae in?vivo, which is due to impaired transport of caveolin-1-containing vesicles along microtubules (MT) to the plasma membrane. Mechanistically, ILK promotes the recruitment of the F-actin binding protein IQGAP1 to the cell cortex, which, in turn, cooperates with its?effector mDia1 to locally stabilize MTs and to allow?stable insertion of caveolae into the plasma membrane. Our results assign an important role to the integrin/ILK complex for caveolar trafficking to the cell surface.     

Incidence of Virulence Factors and Antibiotic Resistance among Enterococci Isolated from Food

The incidence of virulence factors among 48 Enterococcus faecium and 47 Enterococcus faecalis strains from foods and their antibiotic susceptibility were investigated. No strain was resistant to all antibiotics, and for some strains, multiple resistances were observed. Of E. faecium strains, 10.4% were positive for one or more virulence determinants, compared to 78.7% of E. faecalis strains. Strains exhibiting virulence traits were not necessarily positive for all traits; thus, the incidence of virulence factors may be considered to be strain specific.     

Proteome dynamics during plastid differentiation in rice

We have analyzed proteome dynamics during light-induced development of rice (Oryza sativa) chloroplasts from etioplasts using quantitative two-dimensional gel electrophoresis and tandem mass spectrometry protein identification. In the dark, the etioplast allocates the main proportion of total protein mass to carbohydrate and amino acid metabolism and a surprisingly high number of proteins to the regulation and expression of plastid genes. Chaperones, proteins for photosynthetic energy metabolism, and enzymes of the tetrapyrrole pathway were identified among the most abundant etioplast proteins. The detection of 13 N-terminal acetylated peptides allowed us to map the exact localization of the transit peptide cleavage site, demonstrating good agreement with the prediction for most proteins. Based on the quantitative etioplast proteome map, we examined early light-induced changes during chloroplast development. The transition from heterotrophic metabolism to photosynthesis-supported autotrophic metabolism was already detectable 2 h after illumination and affected most essential metabolic modules. Enzymes in carbohydrate metabolism, photosynthesis, and gene expression were up-regulated, whereas enzymes in amino acid and fatty acid metabolism were significantly decreased in relative abundance. Enzymes involved in nucleotide metabolism, tetrapyrrole biosynthesis, and redox regulation remained unchanged. Phosphoprotein-specific staining at different time points during chloroplast development revealed light-induced phosphorylation of a nuclear-encoded plastid RNA-binding protein, consistent with changes in plastid RNA metabolism. Quantitative information about all identified proteins and their regulation by light is available in plprot, the plastid proteome database (http://www.plprot.ethz.ch).     

Evaluation of filter paper as a means of transporting inactivated Gram-negative non-fermentative bacteria and Haemophilus spp. for identification using the MALDI-TOF MS system

This study aimed to evaluate the filter paper as a means to transport inactivated Gram-negative non-fermentative (GNNF) bacteria and Haemophilus spp. for analysis using MALDI-TOF MS. A total of 133 isolates were evaluated and the analysis of each isolate was performed directly from original bacterial colony and in filter paper after the processing. To evaluate the agreement between the identification performed directly from the colony and after impregnation in filter paper, we assign the scores: >2·3 as excellent (E); 2·0 to 2·3 as very good (VG); 1·7–1·99 as good (G); <1·7 as unidentified (U). The divergences were classified as: Minor Divergence, Intermediate Divergence and Major Divergence. A total of 80 isolates transported in the filter paper disks presented full category concordance; 39 isolates presented Minor Divergence; 4 isolates present Intermediate Divergence; 4 isolates present Major Divergence and 6 isolates present better results after impregnation in filter paper. The proposed methodology of bacteria transportation presented a sensitivity of 96·9% and a specificity of 100%. The filter paper as a means to transport and storage of inactivated GNNF and Haemophilus spp. may be considered a potential tool for faster, more accurate, biosafe and less-expensive identification.     

FTIR spectra of algal species can be used as physiological fingerprints to assess their actual growth potential

Fourier transform infrared (FTIR) spectra were measured from cells of Microcystis aeruginosa and Protoceratium reticulatum, whose growth rates were manipulated by the availability of nutrients or light. As expected, the macromolecular composition changed in response to the treatments. These changes were species‐specific and depended on the type of perturbation applied to the growth regime. Microcystis aeruginosa showed an increase in the carbohydrate‐to‐protein ratio with decreased growth rates, under nutrient limitation, whereas light limitation induced a decrease of the carbohydrate‐to‐protein ratio with decreasing proliferation rates. The macromolecular pools of P. reticulatum showed a higher degree of compositional homeostasis. Only when the lowest light irradiance and nutrient availability were supplied, an increase of the carbohydrate‐to‐protein FTIR absorbance ratio was observed. A species‐specific partial least squares (PLS) model was developed using the whole FTIR spectra. This model afforded a very high correlation between the predicted and the measured growth rates, regardless of the growth conditions. On the contrary, the prediction based on absorption band ratios generally used in FTIR studies would strongly depend on growth conditions. This new computational method could constitute a substantial improvement in the early warning systems of algal blooms and, in general, for the study of algal growth, e.g. in biotechnology. Furthermore, these results confirm the suitability of FTIR spectroscopy as a tool to map complex biological processes like growth under different environmental conditions.     

Individualgeschichte und Gesangsauspr?gung beim Star (Sturnus vulgaris) in einer Kolonie

Zusammenfassung Die Lautäußerungen der Stare können in Gesangsstrophen und Pfeiflaute unterteilt werden. Pfeiflaute sind kurz (bis 2 s) und einfach gebaut, während die Strophen wesentlich länger andauern (meist über 20 s) und sich aus zahlreichen komplex gebauten Untereinheiten zusammensetzen. In einer Kolonie bei Frankfurt am Main, in der die Individuen durch Farbringe gekennzeichnet sind, wurde der individuelle Variationsspielraum der Lautäußerungen analysiert. In den Feinstrukturen der Pfeifthemen glichen sich die Koloniemitglieder weitgehend aneinander an. In den Feinstrukturen der Motivtypen der Gesangsstrophen traten dagegen große individuelle Unterschiede auf. , die während Jahren in der gleichen Kolonie brüteten, sangen nur sehr wenige gemeinsame Motivtypen. Die Repertoiregröße variierte zwischen 17–39 Motivtypen. Im Herbstgesang traten 5 bisher noch nicht beobachtete Motivtypen auf. Es gibt somit deutliche Hinweise darauf, daß auch der adulte Star neue Laute erlernen kann.

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Individual history and song structure of Starlings (Sturnus vulgaris) living in a colony

Summary Starling's song can be divided into warbling and whistles. Whistles have a short and simple structure, whereas warbling is sung in long sequences (most often more than 20 s). In the warbling one can distinguish many different subunits (repetition of motifs). The individual variability of songs was studied in a colony near Frankfurt/Main in West Germany, where starlings were colour banded since 1969. The following results were obtained:Birds from the same colony uttered nearly identical or very similar microstructures in the species-specific whistle themes. Different to the whistle themes very distinct individual differences characterized the warbling. Only a few motif types were common among colony members. The individual repertoire size ranged from 17 to 39 motif types. Comparison of the song of one individual between the breeding season and the following autumn shows that motifs, which were not observed during spring occurred in the autumn. This observation is a hint that starlings are capable to learn new motif types even as adults.

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Stipendium der Alexander-von-Humboldt-Stiftung