Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer¹. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells^2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/α-ketoglutarate-dependent dioxygenases^{2, 3}. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development^{2, 5-10}. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC¹¹. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically¹².Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by β-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.     

Identification and gene expression profiling of the Pum1 and Pum2 members of the Pumilio family in the chicken

Members of the Pumilio (Pum) family of RNA-binding proteins act as translational repressors and are required for germ cell development and asymmetric division. We identified the chicken Pum1 and Pum2 genes and analyzed their expression patterns in various tissues. Comparative sequence analysis of the Pum1 and Pum2 proteins from the drosophila, chicken, mouse, and human revealed a high degree of evolutionary conservation in terms of the levels of homology of the peptide sequences and the structure of Pumilio homology domain (PUM-HD), C-terminal RNA-binding domain, with similar spacing between the adjacent Pum eight tandem repeats. In addition, phylogenetic patterns of pumilio family showed that Pum 1 and 2 of chicken are more closely related to those of mouse and human than other species and Pum1 is more conserved than Pum2. Using real-time RT-PCR, the expression levels of the Pum1 and Pum2 genes were found to be highest in hatched female gonads, and high-level expression of Pum2 was detected in 12-day and hatched gonads among the various chicken embryonic tissues tested. In adult tissues, the expression levels of Pum1 and Pum2 were expressed at higher levels in the testis and muscle than in any other tissue. The characteristics of the tissue-specific expression of Pum genes suggest that Pum1 and Pum2 have effects crucially in particular stage during development of chicken gonads depending on sexual maturation.     

Proteomics-based target identification: bengamides as a new class of methionine aminopeptidase inhibitors

LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3gamma, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3gamma. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.     

Central beta-amyloid peptide-induced peripheral interleukin-6 responses in mice

beta-Amyloid peptides (Abetas) share with lipopolysaccharide, a potent pro-inflammatory agent, the property of stimulating glial cells or macrophages to induce various inflammatory mediators. We recently reported that central administration of lipopolysaccharide induces peripheral interleukin-6 responses via both the central and peripheral norepinephrine system. In this study, the effect of intracerebroventricular injection of various synthetic Abetas on plasma interleukin-6 levels was examined in mice. Abeta(1-42) dose-dependently increased plasma interleukin-6 levels: 'aged' Abeta(1-42) was more effective than fresh, whereas Abeta(42-1) had no effect. 'Aged' Abeta(1-42) (205 pmol/mouse i.c.v.)-induced plasma interleukin-6 peaked at 2 h post injection, which is earlier than the peak time of the Abeta(1-42)-induced brain interleukin-6, tumor necrosis factor-alpha and interleukin-1beta levels, which was 4, 4 and 24 h, respectively. Among various peripheral organs, Abeta(1-42) (205 pmol/mouse i.c.v.) significantly increased interleukin-6 mRNA expression in lymph nodes and liver. Abeta(1-42) (205 pmol/mouse i.c.v.) significantly increased norepinephrine turnover in both hypothalamus and spleen. Either central or peripheral norepinephrine depletion effectively inhibited the Abeta(1-42)-induced peripheral interleukin-6 response. Pretreatment with prazosin (alpha(1)-adrenergic antagonist), yohimbine (alpha(2)-adrenergic antagonist), and ICI-118,551 (beta(2)-adrenergic antagonist), but not with betaxolol (beta(1)-adrenergic antagonist), inhibited Abeta(1-42)-induced plasma interleukin-6 levels. These results demonstrate that centrally administered Abeta(1-42) effectively induces the systemic interleukin-6 response which is mediated, in part, by central Abeta(1-42)-induced activation of the central and the peripheral norepinephrine systems.     

Dibenzocyclooctadiene-type lignans from Magnolia pyramidata

Eight dibenzocyclooctadiene-type lignans, pyramidatin A-H, were isolated from the leaves of Magnolia pyramidata. Their structures were established by spectral methods, mainly 2D NMR spectroscopic techniques, which involved combined applications of COSY, DEPT. 1H, 13C correlations, COLOC, INAPT and long-range inverse 1H, 13C NMR correlations. The molecular structures of pyramidatin A and B were determined by single crystal X-ray diffraction. The absolute configurations of all eight lignans were derived from CD spectral correlations with structurally related dibenzocyclooctadienes of known absolute configuration.     

Inhibition of fatty acid synthase suppresses U-2 OS cell invasion and migration via downregulating the activity of HER2/PI3K/AKT signaling pathway in vitro

FASN plays an important role in the malignant phenotype of various tumors. Our previous studies show that inhibition FASN could induce apoptosis and inhibit proliferation in human osteosarcoma (OS) cell in vivo and vitro. The aim in this study was to investigate the effect of inhibition FASN on the activity of HER2/PI3K/AKT axis and invasion and migration of OS cell. The expression of FASN, HER2 and p-HER2(Y1248) proteins was detected by immunohistochemistry in OS tissues from 24 patients with pulmonary metastatic disease, and the relationship between FASN and p-HER2 as well as HER2 was investigated. The results showed that there was a positive correlation between FASN and HER2 as well as p-HER2 protein expression. The U-2 OS cells were transfected with either the FASN specific RNAi plasmid or the negative control RNAi plasmid. FASN mRNA was measured by RT-PCR. Western blot assays was performed to examine the protein expression of FASN, HER2, p-HER2(Y1248), PI3K, Akt and p-Akt (Ser473). Migration and invasion of cells were investigated by wound healing and transwell invasion assays. The results showed that the activity of HER2/PI3K/AKT signaling pathway was suppressed by inhibiting FASN. Meanwhile, the U-2OS cells migration and invasion were also impaired by inhibiting the activity of FASN/HER2/PI3K/AKT. Our results indicated that inhibition of FASN suppresses OS cell invasion and migration via down-regulation of the “HER2/PI3K/AKT” axis in vitro. FASN blocker may be a new therapeutic strategy in OS management.     

Phytoplankton biomass and production in northern South China Sea during summer: Influenced by Pearl River discharge and coastal upwelling

Chlorophyll a and primary production were studied in northern South China Sea during summer from 2007 to 2008. Microplankton dominated total phytoplankton biomass in the coast, while picoplankton dominated in the offshore. Algae bloom caused by Thalassionema nitzschioides was found at the subsurface of upwelling regions (D2, C2) in 2008, and maximum of phytoplankton abundance reached 1.58 × 10⁶ ind L^?1. Integrated primary production ranged from 189.3 to 976.2 mg m^?2 d^?1 in 2007, and ranged from 652.1 to 6601 mg m^?2 d^?1 in 2008. PP showed positive relationship with IPP (p < 0.01) and negative relationship with SST (p < 0.05). Coastal upwelling and Pearl River discharge sustained high PP, and played important role in regulating the phytoplankton biomass and production.     

Isosteric ramatroban analogs: selective and potent CRTH-2 antagonists

The chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH-2), also found on eosinophils and basophils, is a prostaglandin D2 receptor involved in the recruitment of these cell types during an inflammatory response. In this report, we describe the synthesis and optimization of a ramatroban isostere that is a selective and potent antagonist of CRTH-2 which may be useful in the treatment of certain diseases.