Peony Glycosides Protect Against Corticosterone-Induced Neurotoxicity in PC12 Cells

Preclinical and clinical investigations have shown the involvement of dysregulation of hypothalamic–pituitary–adrenal (HPA) axis in the pathogenesis of depression. Hypercortisolemia and the associated hippocampal atrophy were observed in patients with depression, which could be ameliorated by the treatment with antidepressants. Therefore, neuroprotection has been proposed to be one of the acting mechanisms of antidepressant. Previous studies in our laboratory have demonstrated the antidepressant-like activity of total glycosides of peony (TGP) in mice. This study aimed to examine the effect of TGP treatment on corticosterone-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Treating the cells with corticosterone at 200 μM for 48 h caused apoptotic cell death. The cytotoxicity was associated with the activation of caspase-3 activity and the decrease in the mRNA ratio of bcl-2 to bax. TPG treatment at increasing doses (1–10 mg/l) protected against the corticosterone-induced toxicity in PC12 cells in a dose-dependent manner. The cytoprotection afforded by TGP treatment was associated with the inhibition of caspase-3 activity and the up-regulation of bcl-2/bax mRNA ratio. The anti-apoptotic effect of TGP is therefore likely mediated by the suppression of the mitochondrial pathway leading to apoptosis.     

Labeled microRNA pull-down assay system: an experimental approach for high-throughput identification of microRNA-target mRNAs

We developed a simple, direct and cost-effective approach to search for the most likely target genes of a known microRNA (miRNA) in vitro. We term this method ‘labeled miRNA pull-down (LAMP)’ assay system. Briefly, the pre-miRNA is labeled with digoxigenin (DIG), mixed with cell extracts and immunoprecipitated by anti-DIG antiserum. When the DIG-labeled miRNA and bound mRNA complex are obtained, the total cDNAs are then subcloned and sequenced, or RT–PCR-amplified, to search for the putative target genes of a known miRNA. After successfully identifying the known target genes of Caenorhabditis elegans miRNAs lin-4 and let-7 and zebrafish let-7, we applied LAMP to find the unknown target gene of zebrafish miR-1, which resulted in the identification of hand2. We then confirmed hand2 as a novel target gene of miR-1 by whole-mount in situ hybridization and luciferase reporter gene assay. We further validated this target gene by microarray analysis, and the results showed that hand2 is the top-scoring among 302 predicted putative target genes. We concluded that LAMP is an experimental approach for high-throughput identification of the target gene of known miRNAs from both C. elegans and zebrafish, yielding fewer false positive results than those produced by using only the bioinformatics approach.     

Characterization of fungal soil communities by F-RISA and arbuscular mycorrhizal fungi fromAraucaria angustifolia forest soils after replanting and wildfire disturbances

The Fungal Ribosomallntergenic Spacer Analysis (F-RlSA) was used to characterize soil fungal communities from three Cecosystems ofAraucaria angustifolia from Brazil: a native forest and two replanted forest ecosystems, one of them with a past history of wildfire. The arbuscular mycorrhizal fungi (AMF) infection was evaluated inAraucaria roots of 18-monthold axenic plants previously inoculated with soils collected from those areas in a greenhouse experiment. The principal componentanalysis of F-RISA profiles showed different soil fungal community betweenthe three studied areas. Sixty three percent of F-RISA fragments amplified in the soil and the substrate samples presented lengths between 500 and 700 bp. The number of Operational Taxonomic Units (OTUs) was 34 for soil and 38 for substrate, however, more fragments were detected in soil (214) than in substrate (163). Anin silico F-RISA analysis to compare our data with ITSI-5.8S-ITS2 sequences from NCBI database showed the presence of Ascomycota, Basidiomycota and Glomeromycota among the soil and substrate fungal communities. AMF infection was higher in plants inoculated with soil from the native forest and the replanted forest with wildfire, both presenting similar chemical characteristics but with different disturbance levels. These results indicate that soil chemical composition may influence the soil fungal community structures rather than the anthropogenicor fire disturbances.     

Changes in the Mark–Houwink hydrodynamic volume of chitosan molecules in solutions of different organic acids, at different temperatures and ionic strengths

The objective of this study is to explore the cause(s) of changes in the hydrodynamic volume of chitosan molecules in solutions of different organic acids, at different temperatures and ionic strengths. Change in intrinsic viscosity is used as the parameter to elucidate the causes of changes in the hydrodynamic volume of chitosan molecules in these solutions. Results show that the intrinsic viscosity of chitosan decreases in acetic acid or in malic acid over storage time. These decreases are more pronounced in acetic acid solution than in malic acid solution, more significant in higher temperature than in lower temperature solutions, and greater in solutions without NaCl than in solutions containing higher NaCl. The decrease in intrinsic viscosity can perhaps be attributed to the compounded effects of compaction of the chitosan molecules and/or acidic degradation during storage.     

Glucosamine inhibits IL‐1β‐mediated IL‐8 production in prostate cancer cells by MAPK attenuation

Inflammation is a complex process involving cytokine production to regulate host defense cascades. In contrast to the therapeutic significance of acute inflammation, a pathogenic impact of chronic inflammation on cancer development has been proposed. Upregulation of inflammatory cytokines, such as IL‐1β and IL‐8, has been noted in prostate cancer patients and IL‐8 has been shown to promote prostate cancer cell proliferation and migration; however, it is not clear whether IL‐1β regulates IL‐8 expression in prostate cancer cells. Glucosamine is widely regarded as an anti‐inflammatory agent and thus we hypothesized that if IL‐1β activated IL‐8 production in prostate cancer cells, then glucosamine ought to blunt such an effect. Three prostate cancer cell lines, DU‐145, PC‐3, and LNCaP, were used to evaluate the effects of IL‐1β and glucosamine on IL‐8 expression using ELISA and RT‐PCR analyses. IL‐1β elevated IL‐8 mRNA expression and subsequent IL‐8 secretion. Glucosamine significantly inhibited IL‐1β‐induced IL‐8 secretion. IL‐8 appeared to induce LNCaP cell proliferation by MTT assay; involvement of IL‐8 in IL‐1β‐dependent PC‐3 cell migration was demonstrated by wound‐healing and transwell migration assays. Inhibitors of MAPKs and NFκB were used to pinpoint MAPKs but not NFκB being involved in IL‐1β‐mediated IL‐8 production. IL‐1β‐provoked phosphorylation of all MAPKs was notably suppressed by glucosamine. We suggest that IL‐1β can activate the MAPK pathways resulting in an induction of IL‐8 production, which promotes prostate cancer cell proliferation and migration. In this context, glucosamine appears to inhibit IL‐1β‐mediated activation of MAPKs and therefore reduces IL‐8 production; this, in turn, attenuates cell proliferation/migration. J. Cell. Biochem. 108: 489–498, 2009. © 2009 Wiley‐Liss, Inc.     

Baicalein induces proliferation inhibition in B16F10 melanoma cells by generating reactive oxygen species via 12-lipoxygenase

In a previous study, we demonstrated that baicalein induces hydroxyl radical formation in human platelets but the mechanisms are unclear. Herein, we show, using an electron spin resonance technique, that baicalein also induces hydroxyl radical formation in B16F10 melanoma cells in a dose-dependent manner. Baicalein produced superoxide anions in the presence of an iron chelator and superoxide dismutase (SOD) inhibitor. We suggest that superoxide anions produced by baicalein were promptly converted to hydroxyl radicals through SOD and the Fenton reaction in B16F10 melanoma cells. According to Western blotting results, the 12-LOX protein was expressed in B16F10 melanoma cells, but baicalein had no effect on 12-LOX expression. Decreases in 12-LOX protein expression and hydroxyl radical signals occurred in a 12-LOX small interfering RNA knockdown protein group compared with the baicalein control. In the MTT assay, we also found that baicalein caused a reduction in cellular viability, which was reversed by the addition of ROS scavengers. On the basis of these data, we conclude that ROS formation catalyzed by 12-LOX is one possible mechanism of growth inhibition by baicalein in B16F10 melanoma cells.     

Identification of Coffee Genes Expressed During Systemic Acquired Resistance and Incompatible Interaction with Hemileia vastatrix

Coffee genes associated with systemic acquired resistance (SAR) and incompatible reaction against coffee leaf rust inoculation were identified by suppression subtractive hybridization. Analysis of 384 clones of each of the subtracted cDNA libraries identified genes involved in oxidative burst/apoptosis/hypersensitive response, synthesis of antimicrobial proteins, synthesis and transport of antimicrobial metabolites, signal perception and transduction, metabolism of lipids, regulated protein degradation and cell maintenance and development. Induction of distinct sets of genes in the two resistance responses was observed. A wide range of genes involved in defence responses described in other plant species was also found in coffee plants. Semi-quantitative and quantitative RT-PCR analysis of seven selected genes showed differences in their expression profile within 72 h after treatment. Full-length cDNA sequences of two β-1,3-glucanases, one induced during SAR and the other in the incompatible reaction, were obtained by 5' and 3' RACE and the sequence data suggest different properties and cellular localization of the encoded proteins.