Negative regulation of beta-catenin/Tcf signaling by naringenin in AGS gastric cancer cell

Functional activation of beta-catenin/Tcf signaling plays an important role in early events in carcinogenesis. We examined the effect of naringenin against beta-catenin/Tcf signaling in gastric cancer cells. Reporter gene assay showed that naringenin inhibited beta-catenin/Tcf signaling efficiently. In addition, the inhibition of beta-catenin/Tcf signaling by naringenin in HEK293 cells transiently transfected with constitutively mutant beta-catenin gene, whose product is not phosphorylated by GSK3beta, indicates that its inhibitory mechanism was related to beta-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunofluorescence, Western blot, and EMSA. As a result, our data revealed that the beta-catenin distribution and the levels of nuclear beta-catenin and Tcf-4 proteins were unchanged after naringenin treatment. Moreover, the binding activities of Tcf complexes to consensus DNA were not affected by naringenin. Taken together, these data suggest that naringenin inhibits beta-catenin/Tcf signaling in gastric cancer with unknown mechanisms.     

Characterization of the human GARP (Golgi associated retrograde protein) complex

The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the "orphan" SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells.     

Hydrogen-bonded supramolecular manganese(II) complexes with network and sheet structures bridged by terephthalate unit

Two 1D complexes [Mn(4- methylpyrazole)₃(H₂O)(tp)]_n (2) and [Mn(4-methylpyrazole)₄(tp)]_n (3) (tp = terephthalate) were synthesized and characterized by means of X-ray analysis and magnetic studies. The molecular structure of 2 reveals that Mn(II) centers with asymmetric coordination surroundings are bridged by crystallographically different tp ligands, forming a 1D chain. The 1D coordination chains are interconnected by hydrogen bonds between free carboxylate oxygen atoms in a chain and hydrogens of pyrazole nitrogen atoms in neighboring chains, leading to a 3D framework. Compound 3 also exhibits a 1D coordination chain which is hydrogen-bonded to adjacent chains, providing a 2D sheet structure. Interestingly, the structures include intra- and interchain hydrogen bonds contributed from N-H groups of the capping 4-methylpyrazole ligands. Magnetic measurements show weak antiferromagnetic interactions with exchange coupling parameters of J = −0.018 cm⁻¹ for 2 and J = −0.062 cm⁻¹ for 3 through the extended tp ligand on the basis of an infinite chain model (H = −J∑S_i · S_i + 1).     

Changes in pathogenesis-related proteins in pepper plants with regard to biological control of phytophthora blight with <Emphasis Type="Italic">Paenibacillus illinoisensis</Emphasis>

To evaluate the biocontrol effectiveness of chitinase-producing bacterium, Paenibacillus illinoisensis strain KJA-424 against pathogenic strain of Phytophthora capsici in pepper plants, growth response and kinetics of pathogen related (PR) proteins were estimated after inoculation with P. capsici (P), and with a combination of P. capsici and strain KJA-424 cell culture (P+A). Fresh weight and chlorophyll content in shoots at P+A-treated plants significantly increased by 23.4 and 34.2%, respectively after 7days of inoculation, compared to P-treated plants. Root mortality in P+A-treated plants was significantly reduced compared to P-treated plants. Seven days after inoculation, the activities of -1,3-glucanase, cellulase and chitinase in P-treated roots had decreased by 54.8, 36.5 and 52.8%, respectively, compared to P+A-treated roots, while those in P-treated leaves increased by 22.8, 36.3 and 23.8%, respectively, compared to those in P+A-treated leaves. The activities of -1,3-glucanase, cellulase and chitinase in roots are negatively correlated with root mortality. All these results suggest that the inoculation of an antagonist, P. illinoisensis alleviates root mortality, reduction of PR proteins in roots, and activates of PR proteins in leaves infected by P. capsici.     

New insights into metabolic properties of marine bacteria encoding proteorhodopsins

Proteorhodopsin phototrophy was recently discovered in oceanic surface waters. In an effort to characterize uncultured proteorhodopsin-exploiting bacteria, large-insert bacterial artificial chromosome (BAC) libraries from the Mediterranean Sea and Red Sea were analyzed. Fifty-five BACs carried diverse proteorhodopsin genes, and we confirmed the function of five. We calculate that proteorhodopsin-exploiting bacteria account for 13% of microorganisms in the photic zone. We further show that some proteorhodopsin-containing bacteria possess a retinal biosynthetic pathway and a reverse sulfite reductase operon, employed by prokaryotes oxidizing sulfur compounds. Thus, these novel phototrophs are an unexpectedly large and metabolically diverse component of the marine microbial surface water.     

Characterization of N alpha-acetyl methionyl human growth hormone formed during expression in Saccharomyces cerevisiae with liquid chromatography and mass spectrometry

We found a new variant of human growth hormone (hGH) from the recombinant hGH expression process in Saccharomyces cerevisiae. The variant was identified as N(alpha)-acetyl methionyl hGH which may be formed by N(alpha)-acetylation of met-hGH during the intracellular expression of hGH in S. cerevisiae. The variant was isolated from manufacturing process of LG Life Sciences' hGH product. The variant was subjected to trypsin digestion and RP-HPLC analysis, resulting in a delayed retention time and an increased mass (173 Da) of T1 tryptic peptide. The amino acid composition and amino acid sequence of the peptide showed the same result with T1 peptide of met-hGH except the N-terminal modification on methionine in the variant peptide. With collision induced dissociation (CID) experiments of the variant T1 tryptic peptide, we found the sequence and the a(1) fragment of N-terminal residue matched with those of acetyl-methionyl hGH. Within our production process, we produce the methionyl hGH first and then use the aminopeptidase to cut the N-terminal methionine. So the acetylation may inhibit the aminopeptidase to remove methionine and produces N(alpha)-acetyl methionyl hGH. And the biological activity of the variant was comparable to one of the unmodified hGH when tested by rat weight gain bioassay.     

Eutectic formation analysis of amino acid mixtures using molecular dynamics simulations

The mechanism of eutectic formation was investigated via computer-aided molecular dynamics techniques based on experimental results. The CBZ group mixtures CBZ-l-Asp/d-AlaNH2 x HCl/methanol, CBZ-l-Asp/l-PheOMe x HCl/methanol, and CBZ-l-Tyr/l-ArgNH2 x 2HCl/methanol formed transparent eutectic melts. The non-CBZ group mixtures l-Asp/d-AlaNH2 x HCl/methanol, l-Asp/l-PheOMe x HCl/methanol, and l-Tyr/l-ArgNH2 x 2HCl/methanol did not form eutectic melts. According to molecular dynamics simulation results, increase in the kinetic energy values of eutectic forming mixtures was much larger than the increase in potential energy over a temperature shift from 298 to 333 K. However, the results for non-eutectic forming mixtures were reversed. The Coulomb interaction energies of eutectic forming mixtures significantly decreased, because eutectic melting can increase the mobility of molecules in the mixtures. The enhancement of molecular mobility was confirmed by increased self-diffusion constant values, and the change of solid-to-liquid phase was detected by radial distribution function results. The periodic boundary conditions for calculation of molecular dynamics were found to be reliable.     

Activation of the Trk signaling pathway by extracellular zinc. Role of metalloproteinases

In certain brain regions, extracellular zinc concentrations can rise precipitously as intense neuronal activity releases large amounts of zinc from the nerve terminals. Although zinc release has been suggested to play a pathological role, its precise physiological effect is poorly understood. Here, we report that exposure to micromolar quantities of zinc for only a few minutes robustly and specifically activated tropomyosin-related kinase (Trk) receptors, most likely TrkB, in cultured cortical neurons. We further found that Trk activation by zinc is extracellularly mediated by activation of metalloproteinases, which release pro-BDNF from cells and convert pro-BDNF to mature BDNF. These results suggest that activity-dependent release of extracellular zinc leads to metalloproteinase activation, which plays a critically important role in Trk receptor activation at zinc-containing synapses.     

Inactivation of organellar glutamyl- and seryl-tRNA synthetases leads to developmental arrest of chloroplasts and mitochondria in higher plants

Aminoacyl-tRNA synthetases (ARSs) are key enzymes involved in protein translation, and both cytosolic and organellar forms are present in the genomes of eukaryotes. In this study, we investigated cellular effects of depletion of organellar forms of ARS using virus-induced gene silencing (VIGS) in Nicotiana benthamiana. VIGS of NbERS and NbSRS, which encode organellar GluRS and SerRS, respectively, resulted in a severe leaf-yellowing phenotype. The NbERS and NbSRS genes were ubiquitously expressed in plant tissues, and induced in response to light. Green fluorescent protein (GFP) fusion proteins of the full-length glutamyl-tRNA synthetase (ERS) and seryl-tRNA synthetase (SRS) of Arabidopsis and GFP fusions to the N-terminal extension of these proteins were all dualtargeted to chloroplasts and mitochondria. At the cell level, depletion of NbERS and NbSRS resulted in dramatically reduced numbers of chloroplasts with reduced sizes and chlorophyll content. The numbers and/or physiology of mitochondria were also severely affected. The abnormal chloroplasts lacked most of the thylakoid membranes and appeared to be degenerating, whereas some of them showed doublet morphology, indicating defective chloroplast division. Pulse-field gel electrophoresis analyses demonstrated that chloroplast DNA in subgenomic sizes is the predominant form in the abnormal chloroplasts. Interestingly, despite severe abnormalities in chloroplasts and mitochondria, expression of many nuclear genes encoding chloroplastor mitochondria-targeted proteins, and chlorophyll biosynthesis genes remained unchanged in the ERS and SRS VIGS lines. This is the first report to analyze the effect of ARS disruption on organelle development in plants.