Apoptosis induced by troglitazone is both peroxisome proliferator-activated receptor-gamma- and ERK-dependent in human non-small lung cancer cells

The role of the peroxisome proliferator-activated receptor-gamma (PPARgamma) in cell differentiation, cell-cycle arrest, and apoptosis has attracted increasing attention. We have recently demonstrated that PPARgamma ligands-troglitazone (TGZ) induced apoptosis in lung cancer cells. In this report, we further studied the role of ERK1/2 in lung cancer cells treated by TGZ. The result demonstrated that TGZ induced PPARgamma and ERK1/2 accumulation in the nucleus, in which the co-localization of both proteins was found. The activation of ERK1/2 resulted in apoptosis via a mitochondrial pathway. Both PPARgamma siRNA and U0126, a specific inhibitor of ERK1/2, were able to block these effects of TGZ, suggesting that apoptosis induced by TGZ was PPARgamma and ERK1/2 dependent. Inhibition of ERK1/2 by U0126 also led to a significant decrease in the level of PPARgamma, indicating a positive cross-talk between PPARgamma and ERK1/2 or an auto-regulatory feedback mechanism to amplify the effect of ERK1/2 on cell growth arrest and apoptosis. In addition to ERK1/2, TGZ also activated Akt. Interestingly, inhibition of ERK1/2 prevented the activation of Akt whereas the suppression of Akt had no effect on ERK1/2, suggesting that Akt was not necessary for TGZ-PPARgamma-ERK pathway. However, the inhibition of Akt promoted the release of cytochrome c, suggesting the activation of Akt may have a negative effect on apoptosis induced by TGZ. In conclusion, our study has demonstrated that TGZ, a synthetic PPARgamma ligand, induced apoptosis in NCI-H23 lung cancer cells via a mitochondrial pathway and this pathway was PPARgamma and ERK1/2 dependent.     

Nuclear magnetic resonance structure-based epitope mapping and modulation of dust mite group 13 allergen as a hypoallergen

IgE-mediated allergic response involves cross-linking of IgE bound on mast cells by specific surface epitopes of allergens. Structural studies on IgE epitopes of allergens are essential in understanding the characteristics of an allergen and for development of specific allergen immunotherapy. We have determined the structure of a group 13 dust mite allergen from Dermatophagoides farinae, Der f 13, using nuclear magnetic resonance. Sequence comparison of Der f 13 with homologous human fatty acid-binding proteins revealed unique surface charged residues on Der f 13 that may be involved in IgE binding and allergenicity. Site-directed mutagenesis and IgE binding assays have confirmed four surface charged residues on opposite sides of the protein that are involved in IgE binding. A triple mutant of Der f 13 (E41A_K63A_K91A) has been generated and found to have significantly reduced IgE binding and histamine release in skin prick tests on patients allergenic to group 13 dust mite allergens. The triple mutant is also able to induce PBMC proliferation in allergic patients with indices similar to those of wild-type Der f 13 and shift the secretion of cytokines from a Th2 to a Th1 pattern. Mouse IgG serum raised using the triple mutant is capable to block the binding of IgE from allergic patients to wild-type Der f 13, indicating potential for the triple mutant as a hypoallergen for specific immunotherapy. Findings in this study imply the importance of surface charged residues on IgE binding and allergenicity of an allergen, as was also demonstrated in other major allergens studied.     

NADPH oxidase and cyclooxygenase mediate the ultraviolet B-induced generation of reactive oxygen species and activation of nuclear factor-kappaB in HaCaT human keratinocytes

The detrimental effects of ultraviolet B (UVB) irradiation have been connected with the enhanced generation of reactive oxygen species (ROS) by UVB. However, the exact source of ROS produced by UVB has not been clearly revealed yet. In this study, we determined the source of ROS production and its role in the UVB-induced activation of nuclear factor (NF)-kappaB in HaCaT human keratinocytes. UVB irradiation generated ROS in a dose-dependent manner, and this was significantly inhibited by diphenylene iodonium (DPI), apocynin (Apo) and neopterine (Neo), inhibitors of the NADPH oxidase, and indomethacin (Indo), a cyclooxygenase (COX) inhibitor, but not by the mitochondrial electron transport inhibitors and other cytosolic enzyme inhibitors. In addition, these inhibitors of the NADPH oxidase and COX significantly blocked the UVB irradiation-induced nuclear translocation of NF-kappaB. These results suggest that the NADPH oxidase and COX may be major sources for the UVB-induced ROS generation, and play an essential role in the activation of NF-kappaB which is involved in the expression of a variety of genes induced by UVB in HaCaT cells. These results further suggest that these enzymes may be good targets for the preventive strategy of UVB-induced skin injury.     

Synthesis, characterization and crystal structures of Cu(II) and Pd(II) complexes of the pentadentate mixed donor macrocycle, [17]aneNO2S2

An aza-oxa-thia macrocycle, 5,14-dioxa-2,17-dithia[6](1,2)benzeno[6](2,6)pyridinophane, L1, the related smaller macrocycle 2,14-dithia-11-oxa-[3](1,2)benzeno[6](2,6)pyridinophane, L2, and the complexes with Pd(II) and Cu(II) of the macrocycle, L1, have been synthesized. The crystal structure of L2 and those of the two metal complexes have been determined. In the complexes, the metal ions adopt exclusively square planar geometry in which the pyridine nitrogen, two sulfurs and one chlorine atom are coordinated and there is no appreciable interaction with the oxygen donors. Thus, the `hard-soft acid-base' principle is illustrated by the behaviour of L1. The structures of both complexes are compared with the previously reported mixed aza-thia macrocycle, 2,5,14,17-tetrathia[6](1,2)benzeno[6](2,6)pyridinophane. The crystal structure of the smaller macrocycle, L2, is also discussed and due to the nature of its smaller cavity, attempts to make complexes with it have not been successful.     

Alpha7-nicotinic acetylcholine receptors mediate an Abeta(1-42)-induced increase in the level of acetylcholinesterase in primary cortical neurones

The beta-amyloid protein (Abeta) is the major protein component of amyloid plaques found in the Alzheimer brain. Although there is a loss of acetylcholinesterase (AChE) from both cholinergic and non-cholinergic neurones in the brain of Alzheimer patients, the level of AChE is increased around amyloid plaques. Previous studies using P19 cells in culture and transgenic mice which overexpress human Abeta have suggested that this increase may be due to a direct action of Abeta on AChE expression in cells adjacent to amyloid plaques. The aim of the present study was to examine the mechanism by which Abeta increases levels of AChE in primary cortical neurones. Abeta1-42 was more potent than Abeta1-40 in its ability to increase AChE in primary cortical neurones. The increase in AChE was unrelated to the toxic effects of the Abeta peptides. The effect of Abeta1-42 on AChE was blocked by inhibitors of alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) as well as by inhibitors of L- or N-type voltage-dependent calcium channels (VDCCs), whereas agonists of alpha7 nAChRs (choline, nicotine) increased the level of AChE. The results demonstrate that the effect of Abeta1-42 on AChE is due to an agonist effect of Abeta1-42 on the alpha7 nAChR.     

Characterization of RNase-like major storage protein from the ginseng root by proteomic approach

The most abundant root proteins of ginseng (Panax ginseng) have been detected and identified by comparative proteome analysis with cultured hairy root of ginseng. Four abundant proteins (28, 26, 21 and 20 kDa) of P. ginseng had isoforms with different pl values on two-dimensional gel electrophoresis (2DE). The results of N-terminal and internal amino acid sequencing, however, showed that all of them originate from a 28 kDa protein, known as ginseng major protein (GMP). The GMP gene was searched for in the expressed sequence tag database of P. ginseng and found to encode a 27.3 kDa protein having 238 amino acid residues. Analysis of the amino acid sequences indicates that GMP exhibits high sequence homology with plant RNases and RNase-like proteins. However, purified GMP had no RNase activity even though it has conserved amino acid residues known to be essential for active sites of RNase. The GMPs present in ginseng main root were not expressed in cultured hairy roots of ginseng. 2DE analysis showed that the amounts of GMPs in main roots change according to seasonal fluctuation. These results suggest that the GMPs are root-specific RNase-like proteins, which function as vegetative storage proteins of ginseng for survival in the natural environment.     

Differential desensitization and internalization of three different bullfrog gonadotropin-releasing hormone receptors

We previously demonstrated the presence of three distinct types of the gonadotropin-releasing hormone receptor (GnRHR) in a bullfrog (denoted bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). The bfGnRHRs exhibited differential tissue distribution and ligand selectivity. In the present study, we demonstrated the desensitization and internalization kinetics of these receptors in both transiently-transfected HEK293 cells and retrovirus-mediated stable cells. The time-course accumulation of the inositol phosphate in response to GnRH revealed that bfGnRHR-1 and -2 were rapidly desensitized, whereas bfGnRHR-3 was slowly desensitized. A comparison of the internalization kinetics revealed the most rapid rate and highest extent of internalization of bfGnRHR-2 among the three receptors. Interestingly, the mechanisms that underlie the receptor internalization appear to differ from each other. Internalization of bfGnRHR-1 was dependent on both dynamin and beta-arrestin, whereas those of bfGnRHR-2 and -3 were dependent on dynamin, but not on arrestin. These results, therefore, suggest that differential regulatory mechanisms for desensitization and internalization of the GnRHR are involved in diverse cellular and physiological responses to GnRH stimulation.     

Proteome Analysis of Light-induced Proteins in Synechocystis sp. PCC 6803: Identification of Proteins Separated by 2D-PAGE Using N-terminal Sequencing and MALDI-TOF MS

The cyanobacterium Synechocystis sp. PCC 6803 is an ideal model organism for the proteome study of light-induced gene expression because the whole genomic sequence has been determined. The soluble proteins extracted from light- and dark-cultured cells were separated by two-dimensional polyacrylamide gel electrophoresis. Light-induced protein spots electroblotted on a polyvinyldiene difluoride membrane were analyzed by N-terminal Edman sequence determination and followed by CyanoBase. The tryptic digests of some proteins were also confirmed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) and MS-Fit search. Interestingly, eight proteins were related to photosynthesis and respiration (RbcS/L, CbbA, Gap2, AtpB, CpcB, PsbO, and PsbU). Four proteins (SodB, DnaK, GroEL2, and Tig) were involved in cellular processes and the functions of another two proteins (rehydrin and membrane protein) were unknown. The proteome analysis by N-terminal Edman sequencing and MALDI-TOF enabled us to characterize one-shot protein profiles expressed under different physiological conditions.