Preparative chromatography for obtaining pure samples of rosaniline,magenta II,and new fuchsine

A simple low pressure liquid chromatographic method is reported that can separate the basic fuchsine homologues, rosaniline, magenta II and new fuchsine from an impure commercial dye. The chromatographic purity of the separated dyes is > 90%. All homologues were obtained in multi-milligram amounts per chromatographic run; precise yields depend on the composition of the starting material and potentially may be greater. This is a useful preparative procedure for generating chromatographically pure samples of basic fuchsine homologues, especially those that cannot be obtained in pure form by direct synthesis.     

CUL4 associates with DDB1 and DET1 and its downregulation affects diverse aspects of development in Arabidopsis thaliana

Cullins are central scaffolding subunits in eukaryotic E3 ligases that facilitate the ubiquitination of target proteins. Arabidopsis contains at least 11 cullin proteins but only a few of them have been assigned biological roles. In this work Arabidopsis cullin 4 is shown to assemble with DDB1, RBX1, DET1 and DDB2 in vitro and in planta. In addition, by using T-DNA insertion and CUL4 antisense lines we demonstrate that corresponding mutants are severely affected in different aspects of development. Reduced CUL4 expression leads to a reduced number of lateral roots, and to abnormal vascular tissue and stomatal development. Furthermore, cul4 mutants display a weak constitutive photomorphogenic phenotype. These results therefore assign an important function to CUL4 during plant development and provide strong evidence that CUL4 assembles together with RBX1 and DDB1 proteins to form a functional E3 ligase in Arabidopsis.     

AMPK-independent induction of autophagy by cytosolic Ca2+ increase

Autophagy is a eukaryotic lysosomal bulk degradation system initiated by cytosolic cargo sequestration in autophagosomes. The Ser/Thr kinase mTOR has been shown to constitute a central role in controlling the initiation of autophagy by integrating multiple nutrient-dependent signaling pathways that crucially involves the activity of PI3K class III to generate the phosphoinositide PI(3)P. Recent reports demonstrate that the increase in cytosolic Ca²⁺ can induce autophagy by inhibition of mTOR via the CaMKK-α/β-mediated activation of AMPK. Here we demonstrate that Ca²⁺ signaling can additionally induce autophagy independently of the Ca²⁺-mediated activation of AMPK. First, by LC3-II protein monitoring in the absence or presence of lysosomal inhibitors we confirm that the elevation of cytosolic Ca²⁺ induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca²⁺-chelation strongly inhibits autophagy in human, mouse and chicken cells. Strikingly, we found that the PI(3)P-binding protein WIPI-1 (Atg18) responds to the increase of cytosolic Ca²⁺ by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca²⁺-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca²⁺ flux modifications. Importantly, using AMPK-α1^?/?α2^?/? MEFs we show that thapsigargin application triggers autophagy in the absence of AMPK and does not involve complete mTOR inhibition, as detected by p70S6K phosphorylation. In addition, STO-609-mediated CaMKK-α/β inhibition decreased the level of thapsigargin-induced autophagy only in AMPK-positive cells. We suggest that apart from reported AMPK-dependent regulation of autophagic degradation, an AMPK-independent pathway triggers Ca²⁺-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3.     

Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction

Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.     

Solid Oxide Fuel Cells: Microstructure of Nanoscaled La0.6Sr0.4CoO3‐δ Cathodes for Intermediate‐Temperature Solid Oxide Fuel Cells (Adv. Energy Mater. 2/2011)

Nanocrystalline La_1‐xSr_xCoO_3‐δ (LSC) thin films with a nominal Sr‐content of x = 0.4 were deposited on Ce_0.9Gd_0.1O_1.95 electrolyte substrates using a low temperature sol‐gel process. The structural and chemical properties of the LSC thin films were studied after thermal treatment, which included a calcination step and a variable, extended annealing time at 700 °C or 800 °C. Transmission electron microscopy combined with selected‐area electron diffraction, energy‐dispersive X‐ray spectrometry, and scanning transmission electron microscopy tomography was applied for the investigation of grain size, porosity, microstructure, and analysis of the local chemical composition and element distribution on the nanoscale. The area specific resistance (ASR) values of the thin film LSC cathodes, which include the lowest ASR value reported so far (ASR_chem = 0.023 Ωcm² at 600 °C) can be interpreted on the basis of the structural and chemical characterization.     

Microstructure of Nanoscaled La0.6Sr0.4CoO3‐δ Cathodes for Intermediate‐Temperature Solid Oxide Fuel Cells

Nanocrystalline La_1‐xSr_xCoO_3‐δ (LSC) thin films with a nominal Sr‐content of x = 0.4 were deposited on Ce_0.9Gd_0.1O_1.95 electrolyte substrates using a low temperature sol‐gel process. The structural and chemical properties of the LSC thin films were studied after thermal treatment, which included a calcination step and a variable, extended annealing time at 700 °C or 800 °C. Transmission electron microscopy combined with selected‐area electron diffraction, energy‐dispersive X‐ray spectrometry, and scanning transmission electron microscopy tomography was applied for the investigation of grain size, porosity, microstructure, and analysis of the local chemical composition and element distribution on the nanoscale. The area specific resistance (ASR) values of the thin film LSC cathodes, which include the lowest ASR value reported so far (ASR_chem = 0.023 Ωcm² at 600 °C) can be interpreted on the basis of the structural and chemical characterization.     

Proteome analysis in cardiovascular pathophysiology using Dahl rat model

Dahl salt-sensitive (DS) and salt-resistant (DR) inbred rat strains represent a well established animal model for cardiovascular research. Upon prolonged administration of high-salt-containing diet, DS rats develop systemic hypertension, and as a consequence they develop left ventricular hypertrophy, followed by heart failure. The aim of this work was to explore whether this animal model is suitable to identify biomarkers that characterize defined stages of cardiac pathophysiological conditions. The work had to be performed in two stages: in the first part proteomic differences that are attributable to the two separate rat lines (DS and DR) had to be established, and in the second part the process of development of heart failure due to feeding the rats with high-salt-containing diet has to be monitored. This work describes the results of the first stage, with the outcome of protein expression profiles of left ventricular tissues of DS and DR rats kept under low salt diet. Substantial extent of quantitative and qualitative expression differences between both strains of Dahl rats in heart tissue was detected. Using Principal Component Analysis, Linear Discriminant Analysis and other statistical means we have established sets of differentially expressed proteins, candidates for further molecular analysis of the heart failure mechanisms.     

DNA methylation changes associated with risk factors in tumors of the upper aerodigestive tract

Cancers of the upper aerodigestive tract (UADT) are common forms of malignancy associated with tobacco and alcohol exposures, although human papillomavirus and nutritional deficiency are also important risk factors. While somatically acquired DNA methylation changes have been associated with UADT cancers, what triggers these events and precise epigenetic targets are poorly understood. In this study, we applied quantitative profiling of DNA methylation states in a panel of cancer-associated genes to a case-control study of UADT cancers. Our analyses revealed a high frequency of aberrant hypermethylation of several genes, including MYOD1, CHRNA3 and MTHFR in UADT tumors, whereas CDKN2A was moderately hypermethylated. Among differentially methylated genes, we identified a new gene (the nicotinic acetycholine receptor gene) as target of aberrant hypermethylation in UADT cancers, suggesting that epigenetic deregulation of nicotinic acetycholine receptors in non-neuronal tissues may promote the development of UADT cancers. Importantly, we found that sex and age is strongly associated with the methylation states, whereas tobacco smoking and alcohol intake may also influence the methylation levels in specific genes. This study identifies aberrant DNA methylation patterns in UADT cancers and suggests a potential mechanism by which environmental factors may deregulate key cellular genes involved in tumor suppression and contribute to UADT cancers.Key words: DNA methylation, upper aerodigestive tract, cancer, risk factors, biomarkers     

The role of the housefly, Musca domestica, in the spread of Aujeszky''s disease (pseudorabies)

Starved houseflies were held over a suspension of Aujeszky's virus (PRV-1) for 24-48 h. One group was rinsed in 70% ethanol to kill virus attached to the body surface. No virus was isolated from this group. For the other group the titre of virus decreased more rapidly on the body surface of flies than in the environment. Model experiments demonstrated that the Aujeszky's virus cannot survive in the body of the housefly but the body surface may be contaminated for a period of time depending on the initial viral titre. Experiments showed that susceptible pigs fed on flies contaminated with Aujeszky's virus may become infected. The quantity of virus (5 x 10(5) pfu ml-1) shed by a single housefly during biting and vomiting on the cornea or abraded skin proved to be sufficient to cause infection in susceptible pigs, rabbits and a lamb. It is possible that houseflies could play a role in transmission of infection within herds. Transmission between herds is much less likely.