Proteomic Analysis of Coprinopsis cinerea under Conditions of Horizontal and Perpendicular Gravity

Coprinopsis cinerea was employed to investigate the fungal response to gravity. Mycelium growth revealed a consistent growth pattern, irrespective of the direction of gravity (i.e., horizontal vs. perpendicular). However, the fruiting body grew in the direction opposite to that of gravity once the primordia had formed. For the proteomic analysis, only curved-stem samples were used. Fifty-one proteins were identified and classified into 13 groups according to function. The major functional groups were hydrolases and transferases (16%), signal transduction (15%), oxidoreductases and isomerases (11%), carbohydrate metabolism (9%), and transport (5%). To the best of our knowledge, this is the first report on a proteomic approach to evaluate the molecular response of C. cinerea to gravity.

Proteomic analysis of novel targets associated with the enhancement of TrkA-induced SK-N-MC cancer cell death caused by NGF

Nerve growth factor (NGF) is known to regulate both cancer cell survival and death signaling, depending on the cellular circumstances, in various cell types. In this study, we showed that NGF strongly upregulated the protein level of tropomyosin-related kinase A (TrkA) in TrkA-inducible SK-N-MC cancer cells, resulting in increases in various TrkA-dependent cellular processes, including the phosphorylation of c-Jun N-terminal kinase (JNK) and caspase-8 cleavage. In addition, NGF enhanced TrkA-induced morphological changes and cell death, and this effect was significantly suppressed by the JNK inhibitor SP600125, but not by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. To investigate novel targets associated with the enhancement of TrkA-induced SK-N-MC cell death caused by NGF, we performed Coomassie Brilliant Blue staining and two-dimensional (2D) proteomic analysis in TrkA-inducible SK-N-MC cells. We identified 31 protein spots that were either greatly upregulated or downregulated by TrkA during NGF treatment using matrix-associated laser desorption/ionization time of flight/time of flight mass spectrometry, and we analyzed the effects of SP600125 and wortmannin on the spots. Interestingly, 11 protein spots, including heterogeneous nuclear ribonucleoprotein K (hnRNP K), lamin B1 and TAR DNA-binding protein (TDP43), were significantly influenced by SP600125, but not by wortmannin. Moreover, the NGF/TrkA-dependent inhibition of cell viability was significantly enhanced by knockdown of hnRNP K using small interfering RNA, demonstrating that hnRNP K is a novel target associated with the regulation of TrkA-dependent SK-N-MC cancer cell death enhanced by NGF.

Genetic introduction of foreign genes to Pleurotus eryngii by restriction enzyme-mediated integration.

Pleurotus eryngii was transformed via restriction enzyme-mediated integration. In order to construct the transformation plasmid, the enhanced cyan fluorescent protein (ECFP) gene was ligated next to the gpd promoter of the plasmid pAN7-1. Transformation was facilitated via the heat treatment of a transformation mixture containing 1 microg of the HindIII-digested plasmid DNA and 10(6) mushroom protoplasts in 40% polyethyleneglycol solution, resulting in 10-40 hygromycin-resistant transformants. Successful transformation was evidenced by PCR, Southern blot, and confocal fluorescence microscopic analyses on the selected transformants. To date, this is the first report on the transformation of P. eryngii by REMI technique.

Identification of Diachea leucopodia on Strawberry from Greenhouse in Korea

We have detected the slime mold, Diachea leucopodia (GNU06-10) in a strawberry greenhouse located in Sancheong-gun, Gyeongnam. Typical fruiting bodies had developed gregariously on the strawberry leaves, petioles, and plant debris on ground soil habitat, and also surprisingly on plastic pipes and a vinyl covering. Field samples were examined via stereomicroscopy, light microscopy, and SEM for the determination of morphological characteristics. Dark-brown to black spores formed gregariously within the stipitate cylindrical sporangium, and were covered by an iridescent peridium, which may be intact at maturity, or may have disintegrated. The upper portion of the peridium generally breaks up to expose the spores, whereas the lower portion was usually persistent. The results of energy dispersive X-ray spectrometer (EDS) analysis showed that lime was present in the stalk and columella but absent from the spores, capillitium, and peridium. The above characteristics confirm its taxonomic position in the genus Diachea. However, this genus is intermediate in character between the Physarales and Stemonitales of the Myxogastromycetidae. Hence, this genus had been classified as a member of the Stemonitales until the mid-1970's, on the basis of its iridescent peridium and noncalcareous capillitial system, similar to Comatricha of the Stemonitaceae. By way of contrast, emphasis on morphological characteristics, most notably the calcareous stalk and typical columella, places Diachea within the order Physarales. The presence of a phaneroplasmodium during the trophic stage and lime deposition in its sporophores, as was confirmed in this work, supported the inclusion of Diachea in the Physarales, and the noncalcareous capillitial system verified its identification as a member of the Didymiaceae. Further characteristics of the species D. leucopodia include the following: phaneroplasmodium, spore globose 7.5 microm in diameter, very minutely roughened; sporangia 500 microm x 1mm, more or less cylindrical, gregarious, stalked 1.2mm; stalk and columella white.

Comparison of Proteome Component of Helicobacter pylori in Different Atmospheric CO2 Concentration

Helicobacter pylori is a spiral, slow growing gram-negative microaerophilic bacterium. It has been shown to be the etiological agent of gastroduodenal diseases, such as chronic gastritis, gastric and duodenal ulcers, and gastric cancer. General culture condition of H. pylori is 5% O2, 10% CO2 and 100% humid atmosphere. We have compared proliferation protein expression profile of H. pylori incubated under normal microaerophilic (10% CO2) and environment stress (4% CO2, 18% CO2) conditions. H. pylori cultured under environment stress displayed coccoid morphology and timedependent decrease in proliferation. We have further compared the protein expression profiles of H. pylori under normal growing and environment stress conditions by a global proteomic analysis, which includes high-resolution 2-DE followed by matrix-assisted laser desorption/ionization time of flight and nanoelectrospray/tandem mass spectrometry. In total, 42 protein spots were found to be up- or down-regulated by more than 2-fold under environment stress conditions. Of the 42 protein spots processed, 27 spots were identified; they represented 19 genes, including 2 kinds of hypothetical proteins.

Enrichment of Low Abundance Proteins of Helicobacter pylori Strain 26695 by the Heparin Chromatography

Low-abundance cellular proteins normally invisible on the standard two-dimensional SDS-polyacrylamide gel electrophoresis (2-DE SDS-PAGE) map must be enriched appropriately in order to be visualized and identified in cells or tissues. We applied proteins of H. pylori strain 26695 to a immobilized heparin-affinity resin, which has an affinity for nucleic acid-binding proteins, protein biosynthesis factors, and growth factors. The whole cell extract of H. pylori strain 26695 was fractionated by the heparin-agarose chromatography, and was analyzed by 2-DE. The 2-DE SDS-PAGE displayed spots after silver staining, which were identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Among the ca. 150 spots that were processed, 79 proteins representing 57 genes were identified. Eleven proteins were determined to be nucleic acid-associated. Eighteen proteins were newly identified in this study, including DNA topoisomerase I. These results may provide guidance for enriching low abundance proteins of H. pylori and contribute to the construction of a master protein map of H. pylori.