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The UNC-51 serine/threonine kinase of C. elegans plays an essential role in axonal elongation, and unc-51 mutants exhibit uncoordinated movements. We have previously identi®ed mouse and human cDNAs encoding UNC-51-like kinase (ULK1). Here we report the identi®cation and characterization of the second murine member of this kinase family, ULK2. Mouse ULK2 cDNA encodes a putative polypeptide of 1033 aa which has an overall 52% and 33% amino acid identity to ULK1 and UNC-51, respectively. ULKs and UNC-51 share a typical domain structure of an amino-terminal kinase domain, a central proline/serine rich (PS) domain, and a carboxy-terminal (C) domain. Northern blot analysis showed that ULK2 mRNA is widely expressed in adult tissues. In situ hybridization analysis indicated that ULK2 mRNA is ubiquitously localized in premature as well as mature neurons in developing nervous system. ULK2 gene was mapped to mouse chromosome 11B1.3 and rat chromosome 10q23 by FISH. HA-tagged ULK2 expressed in COS7 cells had an apparent molecular size of *150 kDa and was autophosphorylated in vitro. Truncation mutants suggested that the autophosphorylation occurs in the PS domain. Although expression of ULK2 failed to rescue unc-51 mutant of C. elegans, a series of ULK2/UNC-51 chimeric kinases revealed that function of the kinase and PS domains are conserved among species, while the C domain acts in a speciesspeci®c manner. These results suggest that ULK2 is involved in a previously uncharacterized signaling pathway in mammalian cells.

Background: Long noncoding RNA (lncRNA) H19 is emerging as a vital regulatory molecule in the progression of different types of cancer and miR-675 is reported to be embedded in H19's first exon. However, their function and specific mechanisms of action have not been fully elucidated. The aim of this study was to identify a novel lncRNA-microRNA-mRNA functional network in gastric cancer. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the relative expression of H19 and miR-675 in normal (GES-1) and gastric cancer cell lines (SGC-7901, SGC-7901/DDP) as well as in tumor tissues. Gain and loss of function approaches were carried out to investigate the potential roles of H19/miR-675 in cell proliferation and apoptosis. Moreover, Fas associated via death domain (FADD) was validated to be the target of miR-675 via luciferase reporter assay. Western blotting was used to evaluate the protein expression of related signaling pathway. Results: In our study H19 and miR-675 were increased in gastric cancer cell lines and tissues. Overexpression of H19 and miR-675 promoted cell proliferation and inhibited cell apoptosis, whereas knockdown of H19 and miR-675 inhibited these effects. By further examining the underlying mechanism, we showed that H19/miR-675 axis inhibited expression of FADD. FADD downregulation subsequently inhibited the caspase cleavage cascades including caspase 8 and caspase 3. Conclusion: Taken together, our results point to a novel regulatory pathway H19/miR-675/ FADD/caspase 8/caspase 3 in gastric cancer which may be potential target for cancer therapy.

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The plant Ophiopogon japonicus (Thunb.) Ker-Gawl. (Liliaceae), widely distributed in South-east Asia, especially in most areas of China,2) and first recorded in "shengnong Bengcaojing" (early in the third century B.C.), has been used in traditional Chinese medicine to treat inflammatory and cardiovascular diseases for thousands of years.3) Chemical studies have shown that this plant mainly includes saponins, polysaccharide and homoisoflavonoidal compounds. [4][5][6][7][8][9][10] As the scientific evidence for its clinical efficacy, we formerly found that the aqueous extract of Ophiopogon japonicus (ROJ-ext) presented remarkable anti-inflammatory activity and ruscogenin and ophiopogonin D are two of its active components, which supported its traditional use in the treatment of various diseases associated with inflammation. 11)Meanwhile, its cardiovascular activities, such as anti-ischaemia, 12) anti-arrhythmic, 13) inhibiting platelets aggregation, 14) protecting endothelium from apoptosis, 15,16) improving microcirculation, and so on have been confirmed in various assay.17-21) Furthermore, we also found that ROJ-ext significantly inhibited venous thrombosis, which is linked with its endothelial cell-protective and anti-adhesive activities. 22)However, its antithrombotic activity has not been evaluated in other animal models and little is known about whether its two anti-inflammatory components showed antithrombotic effects.Therefore, following our previous work, 11) the present study was undertaken to investigate the antithrombotic effects of ROJ-ext and its two major components, ruscogenin and ophiopogonin D, by using several animal models so as to provide further evidence for its clinical use in the treatment of thrombotic diseases. MATERIALS AND METHODS Plant MaterialsThe dried tuber roots of Ophiopogon japonicus were purchased from Nanjing Medical Material Company (Nanjing, Jiangsu, China) and identified as Ophiopogon japonicus (Thunb.) Ker-Gawl. by Professor Boyang Yu, one of the authors. The voucher specimen (BYY020516) was deposited at the Herbarium of China Pharmaceutical University.Extraction and Isolation The aqueous extract of Ophiopogon japonicus (ROJ-ext) and its two components, ruscogenin and ophiopogonin D, were prepared according to the method described previously.11) In brief, the dried tuber roots of Ophiopogon japonicus (10 kg) were extracted with boiling distilled water (2ϫ100 l). The extract was combined and concentrated to dryness in vaccum. The residue was dissolved with water, ethanol added to the final concentration of 75%, and the mixture left overnight. The supernatant was chromatographed on D101 resin column and eluted with water and 70% ethanol. The 70% ethanol elution was collected and concentrated in vacuum to give 45 g of the extract (ROJ-ext). The dosage of this extract was indicated as the powder.ROJ-ext (20 g) was then chromatographed on a silica gel column with chloroform-methanol (5 : 1) as an eluent and gave a dried fraction. Repeated chromatography over a silica gel and Sep...

Plasma low-density lipoprotein-cholesterol (LDL-C) is mainly taken up and cleared by the hepatocellular LDL receptor (LDL-R). LDL-R gene expression is regulated by the sterol regulatory element binding proteins (SREBPs). Previous studies have shown that curcumin reduces plasma LDL-C and has hypolipidemic and anti-atherosclerotic effects. Herein, we investigated the effect of curcumin on LDL-R expression and its molecular mechanism in HepG2 cells. Curcumin increased LDL-R expression (mRNA and protein) and the resultant uptake of DiI-LDL in a dose- and time-dependent manner. Using a GFP reporter system in a transfected HepG2/SRE-GFP cell line, we found that curcumin activated the sterol regulatory element of the LDL-R promoter. In HepG2/Insig2 cells, curcumin reversed the inhibition of LDL-R expression induced by Insig2 overexpression. These data demonstrate that curcumin increases LDL-R protein expression and uptake activity via the SREBPs pathway. These findings contribute to our further understanding of the cholesterol-lowering and anti-atherosclerotic effects of curcumin.

Hydroxyurea is a potent teratogen; free radical scavengers or antioxidants reduce its teratogenicity. Activator Protein-1 (AP-1) and NF-kappaB are redox-sensitive transcription factors with important roles in normal development and the stress response. This study was designed to determine if exposure to teratogenic doses of hydroxyurea induces oxidative stress and alters gene expression by activating these transcription factors. Pregnant mice were treated with saline or hydroxyurea (400, 500, or 600 mg/kg) on gestation day 9 (GD 9) and killed either on GD 9, 0.5, 3, or 6 h after treatment, to assess oxidative stress and transcription factor activities, or on GD 18, to assess fetal development. Exposure to 400 mg/kg hydroxyurea did not affect the progeny, whereas exposure to 500 or 600 mg/kg resulted in dose-dependent increases in fetal resorptions and malformations, including curly tails, abnormal limbs (oligodactyly, hemimelia, and amelia), and short ribs. Hydroxyurea did not induce oxidative stress, as assessed by the ratio of oxidized to reduced glutathione, nor did it alter NF-kappaB DNA binding activity in the GD 9 conceptus. In contrast, exposure to hydroxyurea at any dose increased AP-1 DNA binding activity in embryos and yolk sacs 0.5 or 3 h after treatment. Hydroxyurea-induced c-Fos heterodimer activity in the embryo peaked 3-4-fold above control at 3 h and remained elevated by 6 h; in contrast, the activity of c-Jun dimers was not altered by drug exposure. A dramatic and region-specific increase in c-Fos immunoreactivity was found in hydroxyurea-treated embryos. The induction of AP-1 DNA binding activity by hydroxyurea represents an early, sensitive marker of the embryonic response to insult.