Role of MEK-ERK pathway in sphingosylphosphorylcholine-induced cell death in human adipose tissue-derived mesenchymal stem cells

Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in a variety of cellular responses. In the present study, we demonstrated that treatment of human adipose tissue-derived mesenchymal stem cells (hATSCs) with D-erythro-SPC resulted in apoptosis-like cell death, as demonstrated by decreased cell viability, DNA strand breaks, the increase of sub-G1 fraction, cytochrome c release into cytosol, and activation of caspase-3. In contrast, the exposure of hATSCs to L-threo-SPC did not induce the cell death, suggesting that the SPC-induced cell death was selective for the D-erythro-stereoisomer of SPC. The D-erythro-SPC-induced cell death was prevented by DEVD-CHO, a caspase-3 specific inhibitor, and Z-VAD-FMK, a general caspase inhibitor, suggesting that the SPC-induced cell death of hATSCs occurs through the cytochrome c- and caspase-3-dependent pathways. In addition, D-erythro-SPC treatment stimulated the activation of mitogen-activated protein kinases, such as ERK and c-Jun NH2-terminal protein kinase (JNK), and the D-erythro-SPC-induced cell death was completely prevented by pretreatment with the MEK inhibitor, U0126, but not by pretreatment with the JNK inhibitor, SP600125, and the p38 MAPK inhibitor, SB202190, suggesting a specific involvement of ERK in the D-erythro-SPC-induced cell death. Pretreatment with U0126 attenuated the D-erythro-SPC-induced release of cytochrome c. From these results, we suggest that ERK is involved in the SPC-induced cell death of hATSC through stimulation of the cytochrome c/caspase-3-dependent pathway.     

Auxin-induced reactive oxygen species production requires the activation of phosphatidylinositol 3-kinase

We recently reported that production of reactive oxygen species (ROS) is essential for auxin-induced gravitropic signaling. Here, we investigated the role of phosphatidylinositol 3-kinase and its product, PtdIns(3)P, in auxin-mediated ROS production and the root gravitropic response. Pretreatment with LY294002, an inhibitor of PtdIns 3-kinase activity, blocked auxin-mediated ROS generation, and reduced the sensitivity of root tissue to gravistimulation. The amount of PtdIns(3)P increased in response to auxin, and this effect was abolished by pretreatment with LY294002. In addition, sequestration of PtdIns(3)P by transient expression of the endosome binding domain in protoplasts abrogated IAA-induced ROS accumulation. These results indicate that activation of PtdIns 3-kinase and its product PtdIns(3)P are required for auxin-induced production of ROS and root gravitropism.     

Uronema marinum: identification and biochemical characterization of phosphatidylcholine-hydrolyzing phospholipase C

Phosphatidylcholine (PC)-specific phospholipase D (PC-PLD) and phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) activities have been detected in Uronema marinum. Partial purification of PC-PLC revealed that two distinct forms of PC-PLC (named as mPC-PLC and cPC-PLC) were existed in membrane and cytosol fractions. The two PC-PLC enzymes showed the preferential hydrolyzing activity for PC with specific activity of 50.4 for mPC-PLC and 28.3 pmol/min/mg for cPC-PLC, but did not hydrolyze phosphatidylinositol or phosphatidylethanolamine. However, the biochemical characteristics and physiological roles of both enzymes were somewhat different. mPC-PLC had a pH optimum in the acidic region at around, pH 6.0, and required approximately 0.4 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC had a pH optimum in the neutral region at around, pH 7.0, and required 1.6 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC, but not mPC-PLC, showed a dose-dependent inhibitory effect on the luminal-enhanced chemiluminescence (CL) responses and the viability of zymosan-stimulated phagocytes of olive flounder, indicating that cPC-PLC may contribute to the parasite evasion against the host immune response. Our results suggest that U. marinum contains PC-PLD as well as two enzymatically distinct PC-PLC enzymes, and that mPC-PLC may play a role in the intercellular multiplication of U. marinum and cPC-PLC acts as a virulence factor, serving to actively disrupt the host defense mechanisms.     

Eckol isolated from Ecklonia cava attenuates oxidative stress induced cell damage in lung fibroblast cells

We have investigated the cytoprotective effect of eckol, which was isolated from Ecklonia cava, against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Eckol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, eckol reduced H(2)O(2) induced cell death in V79-4 cells. In addition, eckol inhibited cell damage induced by serum starvation and radiation by scavenging ROS. Eckol was found to increase the activity of catalase and its protein expression. Further, molecular mechanistic study revealed that eckol increased phosphorylation of extracellular signal-regulated kinase and activity of nuclear factor kappa B. Taken together, the results suggest that eckol protects V79-4 cells against oxidative damage by enhancing the cellular antioxidant activity and modulating cellular signal pathway.     

Solution structure and interactions of the Escherichia coli cell division activator protein CedA

CedA is a protein that is postulated to be involved in the regulation of cell division in Escherichia coli and related organisms; however, little biological data about its possible mode of action are available. Here we present a three-dimensional structure of this protein as determined by NMR spectroscopy. The protein is made up of four antiparallel beta-strands, an alpha-helix, and a large unstructured stretch of residues at the N-terminus. It shows structural similarity to a family of DNA-binding proteins which interact with dsDNA via a three-stranded beta-sheet, suggesting that CedA may be a DNA-binding protein. The putative binding surface of CedA is predominantly positively charged with a number of basic residues surrounding a groove largely dominated by aromatic residues. NMR chemical shift perturbations and gel-shift experiments performed with CedA confirm that the protein binds dsDNA, and its interaction is mediated primarily via the beta-sheet.     

The Telomere-Binding Protein Taz1p as a Target for Modification by a SUMO-1 Homologue in Fission Yeast

In fission yeast (Schizosaccharomyces pombe) the homologue of the mammalian SUMO-1 ubiquitin-like modifier is encoded by the pmt3 gene. A two-hybrid screen using the telomere-binding protein Taz1p as bait identified Pmt3p as an interacting factor. In vitro experiments using purified components of the fission yeast Pmt3p modification system demonstrated that Taz1p could be modified directly by Pmt3p. The amino acid sequence of Taz1p contains a close match to the consensus modification site for SUMO-1, and a PEST sequence similar to those found in established SUMO-1 targets. Although previous experiments have identified an increase in telomere length as one consequence of the pmt3– genotype, we could not detect Pmt3p modification of Taz1p in protein extracts made from exponentially growing haploid cells or any effect of Pmt3p on the localization of GFP-Taz1p at discrete foci in the haploid cell nucleus.     

A suppressive oligodeoxynucleotide enhances the efficacy of myelin cocktail/IL-4-tolerizing DNA vaccination and treats autoimmune disease

Targeting pathogenic T cells with Ag-specific tolerizing DNA vaccines encoding autoantigens is a powerful and feasible therapeutic strategy for Th1-mediated autoimmune diseases. However, plasmid DNA contains abundant unmethylated CpG motifs, which induce a strong Th1 immune response. We describe here a novel approach to counteract this undesired side effect of plasmid DNA used for vaccination in Th1-mediated autoimmune diseases. In chronic relapsing experimental autoimmune encephalomyelitis (EAE), combining a myelin cocktail plus IL-4-tolerizing DNA vaccine with a suppressive GpG oligodeoxynucleotide (GpG-ODN) induced a shift of the autoreactive T cell response toward a protective Th2 cytokine pattern. Myelin microarrays demonstrate that tolerizing DNA vaccination plus GpG-ODN further decreased anti-myelin autoantibody epitope spreading and shifted the autoreactive B cell response to a protective IgG1 isotype. Moreover, the addition of GpG-ODN to tolerizing DNA vaccination therapy effectively reduced overall mean disease severity in both the chronic relapsing EAE and chronic progressive EAE mouse models. In conclusion, suppressive GpG-ODN effectively counteracted the undesired CpG-induced inflammatory effect of a tolerizing DNA vaccine in a Th1-mediated autoimmune disease by skewing both the autoaggressive T cell and B cell responses toward a protective Th2 phenotype. These results demonstrate that suppressive GpG-ODN is a simple and highly effective novel therapeutic adjuvant that will boost the efficacy of Ag-specific tolerizing DNA vaccines used for treating Th1-mediated autoimmune diseases.     

Eicosapentaenoic acid inhibits UV-induced MMP-1 expression in human dermal fibroblasts

Ultraviolet (UV) irradiation regulates UV-responsive genes, including matrix metalloproteinases (MMPs). Moreover, UV-induced MMPs cause connective tissue damage and the skin to become wrinkled and aged. Here, we investigated the effect of eicosapentaenoic acid (EPA), a dietary omega-3 fatty acid, on UV-induced MMP-1 expression in human dermal fibroblasts (HDFs). We found that UV radiation increases MMP-1 expression and that this is mediated by p44 and p42 MAP kinase (ERK) and Jun-N-terminal kinase (JNK) activation but not by p38 activation. Pretreatment of HDFs with EPA inhibited UV-induced MMP-1 expression in a dose-dependent manner and also inhibited the UV-induced activation of ERK and JNK by inhibiting ERK kinase (MEK1) and SAPK/ERK kinase 1 (SEK1) activation, respectively. Moreover, inhibition of ERK and JNK by EPA resulted in the decrease of c-Fos expression and c-Jun phosphorylation/expression induced by UV, respectively, which led to the inhibition of UV-induced activator protein-1 DNA binding activity. This inhibitory effect of EPA on MMP-1 was not mediated by an antioxidant effect. We also found that EPA inhibited 12-O-tetradecanoylphorbol-13-acetate- or tumor necrosis factor-alpha-induced MMP-1 expression in HDFs and UV-induced MMP-1 expression in HaCaT cells. In conclusion, our results demonstrate that EPA can inhibit UV-induced MMP-1 expression by inhibiting the MEK1/ERK/c-Fos and SEK1/JNK/c-Jun pathways. Therefore, EPA is a potential agent for the prevention and treatment of skin aging.     

Estimating the potential refolding yield of recombinant proteins expressed as inclusion bodies

Recombinant protein production in bacteria is efficient except that insoluble inclusion bodies form when some gene sequences are expressed. Such proteins must undergo renaturation, which is an inefficient process due to protein aggregation on dilution from concentrated denaturant. In this study, the protein-protein interactions of eight distinct inclusion-body proteins are quantified, in different solution conditions, by measurement of protein second virial coefficients (SVCs). Protein solubility is shown to decrease as the SVC is reduced (i.e., as protein interactions become more attractive). Plots of SVC versus denaturant concentration demonstrate two clear groupings of proteins: a more aggregative group and a group having higher SVC and better solubility. A correlation of the measured SVC with protein molecular weight and hydropathicity, that is able to predict which group each of the eight proteins falls into, is presented. The inclusion of additives known to inhibit aggregation during renaturation improves solubility and increases the SVC of both protein groups. Furthermore, an estimate of maximum refolding yield (or solubility) using high-performance liquid chromatography was obtained for each protein tested, under different environmental conditions, enabling a relationship between "yield" and SVC to be demonstrated. Combined, the results enable an approximate estimation of the maximum refolding yield that is attainable for each of the eight proteins examined, under a selected chemical environment. Although the correlations must be tested with a far larger set of protein sequences, this work represents a significant move beyond empirical approaches for optimizing renaturation conditions. The approach moves toward the ideal of predicting maximum refolding yield using simple bioinformatic metrics that can be estimated from the gene sequence. Such a capability could potentially "screen," in silico, those sequences suitable for expression in bacteria from those that must be expressed in more complex hosts.