The mammalian target of rapamycin (mTOR) signaling pathway is upregulated in the pathogenesis of many cancers. Arachidonic acid (AA) and its metabolites play critical role in the development of breast cancer, but the mechanisms through which AA promotes mammary tumorigenesis and progression are poorly understood. We found that the levels of AA and cytosolic phospholipase A2 (cPLA2) strongly correlated with the signaling activity of mTORC1 and mTORC2 as well as the expression levels of vascular epithelial growth factor (VEGF) in human breast tumor tissues. In cultured breast cancer cells, AA effectively activated both mTOR complex 1 (mTORC1) and mTORC2. Interestingly, AA-stimulated mTORC1 activation was independent of amino acids, phosphatidylinositol 3-kinase (PI3-K) and tuberous sclerosis complex 2 (TSC2), which suggests a novel mechanism for mTORC1 activation. Further studies revealed that AA stimulated mTORC1 activity through destabilization of mTOR-raptor association in ras homolog enriched in brain (Rheb)-dependent mechanism. Moreover, we showed that AA-stimulated cell proliferation and angiogenesis required mTOR activity and that the effect of AA was mediated by lipoxygenase (LOX) but not cyclooxygenase-2 (COX-2). In animal models, AA-enhanced incidences of rat mammary tumorigenesis, tumor weights and angiogenesis were inhibited by rapamycin. Our findings suggest that AA is an effective intracellular stimulus of mTOR and that AA-activated mTOR plays critical roles in angiogenesis and tumorigenesis of breast cancer.Oncogene advance online publication, 20 February 2012; doi:10.1038/onc.2012.47.

To investigate the regulation of 14-3-3 proteins in banana (Musa acuminata L. AAA group, cv. Brazilian) fruit postharvest ripening, four cDNAs encoding 14-3-3 proteins were isolated from banana and designated as Ma-14-3-3a, Ma-14-3-3c, Ma-14-3-3e, and Ma-14-3-3i, respectively. Amino acid sequence alignment showed that the four 14-3-3 proteins shared a highly conserved core structure and variable C-terminal as well as N-terminal regions with 14-3-3 proteins from other plant species. Phylogenetic analysis revealed that the four 14-3-3 genes belong to the non-ε groups. They were differentially and specifically expressed in various tissues. Real-time RT-PCR analysis indicated that these four genes function differentially during banana fruit postharvest ripening. Three genes, Ma-14-3-3a, Ma-14-3-3c, and Ma-14-3-3e, were significantly induced by exogenous ethylene treatment. However, gene function differed in naturally ripened fruits. Ethylene could induce Ma-14-3-3c expression during postharvest ripening, but expression patterns of Ma-14-3-3a and Ma-14-3-3e suggest that these two genes appear to be involved in regulating ethylene biosynthesis during fruit ripening. No obvious relationship emerged between Ma-14-3-3i expression in naturally ripened and 1-MCP (1-methylcyclopropene)-treated fruit groups during fruit ripening. These results indicate that the 14-3-3 proteins might be involved in various regulatory processes of banana fruit ripening. Further studies will mainly focus on revealing the detailed biological mechanisms of these four 14-3-3 genes in regulating banana fruit postharvest ripening.

AIM To investigate whether there were symptom-based tendencies in the Helicobacter pylori (H. pylori) eradication in functional dyspepsia (FD) patients. METHODS A randomized, single-blind, placebo-controlled study of H. pylori eradication for FD was conducted. A total of 195 FD patients with H. pylori infection were divided into two groups: 98 patients in the treatment group were treated with rabeprazole 10 mg twice daily for 2 wk, amoxicillin 1.0 g and clarithromycin 0.5 g twice daily for 1 wk; 97 patients in the placebo group were given placebos as control. Symptoms of FD, such as postprandial fullness, early satiety, nausea, belching, epigastric pain and epigastric burning, were assessed 3 mo after H. pylori eradication. RESULTS By per-protocol analysis in patients with successful H. pylori eradication, higher effective rates of 77.2% and 82% were achieved in the patients with epigastric pain and epigastric burning than those in the placebo group (P < 0.05). The effective rates for postprandial fullness, early satiety, nausea and belching were 46%, 36%, 52.5% and 33.3%, respectively, and there was no significant difference from the placebo group (39.3%, 27.1%, 39.1% and 31.4%)(P > 0.05). In 84 patients who received H. pylori eradication therapy, the effective rates for epigastric pain (73.8%) and epigastric burning (80.7%) were higher than those in the placebo group (P < 0.05). The effective rates for postprandial fullness, early satiety, nausea and belching were 41.4%, 33.3%, 50% and 31.4%, respectively, and did not differ from those in the placebo group (P > 0.05). By intention-to-treat analysis, patients with epigastric pain and epigastric burning in the treatment group achieved higher effective rates of 60.8% and 65.7% than the placebo group (33.3% and 31.8%)(P < 0.05). The effective rates for postprandial fullness, early satiety, nausea and belching were 34.8%, 27.9%, 41.1% and 26.7% respectively in the treatment group, with no significant difference from those in the placebo group (34.8%, 23.9%, 35.3% and 27.1%)(P > 0.05). CONCLUSION The efficacy of H. pylori eradication has symptom-based tendencies in FD patients. It may be effective in the subgroup of FD patients with epigastric pain syndrome.

It has been reported that metformin, a biguanide derivative widely used in type II diabetic patients, has antitumor activities in some cancers by activation of AMP-activated protein kinase (AMPK). But its role in nasopharyngeal carcinoma (NPC) is not known. Here, we reported for the first time that 1-50 mM of metformin in a dose- and time-dependent manner suppressed cell proliferation and colony formation in NPC cell line, C666-1. Further studies revealed that the protein level of cyclin D1 decreased and the percentage of the cells in G0/G1 phase increased by 5 mM metformin treatment. Metformin also induced the phosphorylation of AMPK (T172) in a time-dependent manner. Mammalian target of rapamycin complex 1 (mTORC1), which is negatively regulated by AMPK and plays a central role in cell growth and proliferation, was inhibited by metformin, as manifested by dephosphorylation of its downstream targets 40S ribosomal S6 kinase 1 (S6K1)(T389), the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1)(T37/46) and S6 (S235/236) in C666-1 cells. In a summary, metformin prevents proliferation of C666-1 cells by down-regulating cyclin D1 level and inducing G1 cell cycle arrest. AMPK-mediated inhibition of mTORC1 signaling may be involved in this process.

Anti-diabetic drug metformin has been shown to enhance osteoblasts differentiation and inhibit osteoclast differentiation in vitro and prevent bone loss in ovariectomized (OVX) rats. But the mechanisms through which metformin regulates osteoclastogensis are not known. Osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) are cytokines predominantly secreted by osteoblasts and play critical roles in the differentiation and function of osteoclasts. In this study, we demonstrated that metformin dose-dependently stimulated OPG and reduced RANKL mRNA and protein expression in mouse calvarial osteoblasts and osteoblastic cell line MC3T3-E1. Inhibition of AMP-activated protein kinase (AMPK) and CaM kinase kinase (CaMKK), two targets of metformin, suppressed endogenous and metformin-induced OPG secretion in osteoblasts. Moreover, supernatant of osteoblasts treated with metformin reduced formation of tartrate resistant acid phosphatase (TRAP)-positive multi-nucleated cells in Raw264.7 cells. Most importantly, metformin significantly increased total body bone mineral density, prevented bone loss and decreased TRAP-positive cells in OVX rats proximal tibiae, accompanied with an increase of OPG and decrease of RANKL expression. These in vivo and in vitro studies suggest that metformin reduces RANKL and stimulates OPG expression in osteoblasts, further inhibits osteoclast differentiation and prevents bone loss in OVX rats.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) the etiology of which has not yet been fully clarified. Cytokine interleukin-10 (IL-10) plays a central role in downregulating inflammatory cascade in UC and is likely a candidate for therapeutic intervention. However, its intravenous administration is costly and inconvenient. Therefore, we established a novel IL-10 delivery system by transforming a hIL-10-containing plasmid into B. longum (BL-hIL-10) and investigated its effects on 5% dextran sulfate sodium (DSS)-induced ulcerative colitis in mice and the possible underlying mechanism. Our results show that (1) hIL-10 was expressed and secreted into the culture supernatant of BL-hIL-10 after L-arabinose induction in vitro as examined by Western blot, enzyme-linked immunosorbent assay (ELISA) and RT-PCR;(2) addition of BL-hIL-10 culture supernatant had no cytotoxic effect and morphological alteration, but significantly inhibited the enhancement of proinflammatory cytokines by lipopolysaccharide (LPS) in THP-1 cells;(3) oral administration of BL-hIL-10 alleviated colitis syndrome of the model mice, attenuated colitis-activated NF-κB pathway measured by DNA-binding assay and colitis-elevated expression of proinflammatory cytokines examined with CCK cytotoxic kits, and upregulated CD4+CD25+Foxp3+ Treg in blood and mesenteric lymph nodes measured by flow cytometry. In conclusion, BL-hIL-10 as a novel oral hIL-10 delivery system has been successfully established and oral administration of BL-hIL-10 alleviated inflammatory damage of colonic tissue in the model mice by blocking the colitis-activated NF-κB pathway and upregulating CD4+CD25+Foxp3+ Treg in blood and mesenteric lymph nodes in mice.

HASH(0x2b98962e2180)

A full-length cDNA encoding an ADP-ribosylation factor (ARF) from banana (Musa acuminata) fruit was cloned and named MaArf. It contains an open reading frame encoding a 181-amino-acid polypeptide. Sequence analysis showed that MaArf shared high similarity with ARF of other plant species. The genomic sequence of MaArf was also obtained using polymerase chain reaction (PCR). Sequence analysis showed that MaArf was a split gene containing five exons and four introns in genomic DNA. Reverse-transcriptase PCR was used to analyze the spatial expression of MaArf. The results showed that MaArf was expressed in all the organs examined: root, rhizome, leaf, flower and fruit. Real-time quantitative PCR was used to explore expression patterns of MaArf in postharvest banana. There was differential expression of MaArf associated with ethylene biosynthesis. In naturally ripened banana, expression of MaArf was in accordance with ethylene biosynthesis. However, in 1-methylcyclopropene-treated banana, the expression of MaArf was inhibited and changed little. When treated with ethylene, MaArf expression in banana fruit significantly increased in accordance with ethylene biosynthesis; the peak of MaArf was 3 d after harvest, 11 d earlier than for naturally ripened banana fruits. These results suggest that MaArf is induced by ethylene in regulating postharvest banana ripening. Finally, subcellular localization assays showed the MaArf protein in the cytoplasm.

A specific, sensitive method was developed for the analysis of chlormequat in wheat and soil by high performance chromatography/mass spectrometry. The fortified recoveries of soil were from 75.08% to 96.55%, with RSD 3.34%-15.18%, the limit of detection of the analytical method was 0.05 ng at a signal-to-noise ratio of 3, and the limit of quantification was 0.05, 0.1, 0.5 mg/kg for soil, wheat plants and wheat grain, respectively. The degradation dynamics and final residues of chlormequat in Beijing and Changchun were investigated. The half-life of chlormequat in wheat plants were 3.15 days in Beijing and 4.56 days in Changchun, while the half-life in soil was 3.88 days in Beijing and 4.51 days in Changchun. The final residues of chlormequat in soil were not detectable, and the final residues of chlormequat in wheat grain were below 0.50 mg/kg except for 3.51 mg/kg from high dosage plot of Changchun. The fact that all the final residues were below 5 mg/kg (GB2763 in National standards of the People's Republic of China, maximum residue limits for pesticide in food, Beijing, 2005) suggested that chlormequat could be safely used in wheat crops with the suitable dosage and application.

Objective To investigate effects of K(ATP) opener on the expressions of caspase-12 mRNA and protein, and to explore the role of endoplasmic reticulum (ER) stress pathway in the mechanism of K(ATP) opener protecting against neuronal apoptosis after cerebral ischemia-reperfusion. Methods Two hundred rats were randomly divided into four groups: sham operation group, ischemia-reperfusion group, K(ATP) opener group, and K(ATP) blocker group. The middle cerebral artery occlusion (MCAO) model was established by intraluminal suture occlusion method; neuronal apoptosis was detected by TUNEL staining. The mRNA and protein expressions of caspase-12 were detected by semi-quantitative RT-PCR and immunohistochemical staining, respectively. Results In ischemia-reperfusion group, K(ATP) opener group and K(ATP) blocker group, the number of apoptotic cells and the mRNA and protein expressions of caspase-12 gradually increased following cerebral reperfusion, and reached the peak at 24 h. In K(ATP) opener group, The number of apoptotic cells was significantly less than that in ischemia-reperfusion group and K(ATP) blocker group at 12 h, 24 h, 48 h and 72 h (P< 0.05 or P< 0.01); while the mRNA and protein levels of caspase-12 were significantly less than those in ischemia-reperfusion group and K(ATP) blocker group at all times (P< 0.05 or P< 0.01). There were no differences between the ischemia-reperfusion group and K(ATP) blocker group at each time (P> 0.05). Conclusion K(ATP) opener may protect neurons from apoptosis following the cerebral ischemia-reperfusion by inhibiting ER stress pathway.