Expression and epigenetic regulation of angiogenesis-related factors during dormancy and recurrent growth of ovarian carcinoma

The initiation of angiogenesis can mark the transition from tumor dormancy to active growth and recurrence. Mechanisms that regulate recurrence in human cancers are poorly understood, in part because of the absence of relevant models. The induction of ARHI (DIRAS3) induces dormancy and autophagy in human ovarian cancer xenografts but produces autophagic cell death in culture. The addition of VEGF to cultures maintains the viability of dormant autophagic cancer cells, thereby permitting active growth when ARHI is downregulated, which mimics the “recurrence” of growth in xenografts. Two inducible ovarian cancer cell lines, SKOv3-ARHI and Hey-ARHI, were used. The expression level of angiogenesis factors was evaluated by real-time PCR, immunohistochemistry, immunocytochemistry and western blot; their epigenetic regulation was measured by bisulfite sequencing and chromatin immunoprecipitation. Six of the 15 angiogenesis factors were upregulated in dormant cancer cells (tissue inhibitor of metalloproteinases-3, TIMP3; thrombospondin-1, TSP1; angiopoietin-1; angiopoietin-2; angiopoietin-4; E-cadherin, CDH1). We found that TIMP3 and CDH1 expression was regulated epigenetically and was related inversely to the DNA methylation of their promoters in cell cultures and in xenografts. Increased H3K9 acetylation was associated with higher TIMP3 expression in dormant SKOv3-ARHI cells, while decreased H3K27me3 resulted in the upregulation of TIMP3 in dormant Hey-ARHI cells. Elevated CDH1 expression during dormancy was associated with an increase in both H3K4me3 and H3K9Ac in two cell lines. CpG demethylating agents and/or histone deacetylase inhibitors inhibited the re-growth of dormant cancer cells, which was associated with the re-expression of anti-angiogenic genes. The expression of the anti-angiogenic genes TIMP3 and CDH1 is elevated during dormancy and is reduced during the transition to active growth by changes in DNA methylation and histone modification.     

Solid tumor-targeted infiltrating cytotoxic T lymphocytes retained by a superantigen fusion protein

Successful immune-mediated regression of solid tumors is difficult because of the small number of cytotoxic T lymphocytes (CTLs) that were traffic to the tumor site. Here, the targeting of tumor-specific infiltrating CTLs was dependent on a fusion protein consisting of human epidermal growth factor (EGF) and staphylococcal enterotoxin A (SEA) with the D227A mutation. EGF-SEA strongly restrained the growth of murine solid sarcoma 180 (S180) tumors (control versus EGF-SEA, mean tumor weight: 1.013 versus 0.197 g, difference  = 0.816 g). In mice treated with EGF-SEA, CD4⁺, CD8⁺ and SEA-reactive T lymphocytes were enriched around the EGFR expressing tumor cells. The EGF receptors were potentially phosphorylated by EGF-SEA stimulation and the fusion protein promoted T cells to release the tumoricidal cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Intratumoral CTLs secreted cytolytic pore-forming perforins and granzyme B proteins near the surface of carcinomas, causing the death of many tumor cells. We additionally show that labeled EGF-SEA was directly targeted to the tumor tissue after intravenous (i.v.) injection. The findings demonstrate that antibody-like EGF-SEA plays an important role in arresting CTLs in the solid tumor site and has therapeutic potential as a tumor-targeting agent.     

Synergistic interactions between Glomus mosseae and Bradyrhizobium japonicum in enhancing proton release from nodules and hyphae

Soybean (Glycine max L. Merr.) seedlings were inoculated with Glomus mosseae (GM) and Bradyrhizobium japonicum (BJ) together or separately to study the effect of interactions on net H⁺ effluxes of nodules or extraradical hyphae by in vivo vibrating electrode techniques. GM promoted three-fold the H⁺ effluxes of nodules on mycorrhizal lateral roots and BJ increased eight-fold the net H⁺ effluxes of hyphae developing in the vicinity of nodules on lateral roots. Increments in plant P content were positively and linearly correlated with the net H⁺ efflux of nodules and hyphae. It is concluded that increased H⁺ effluxes of nodules resulted from enhanced nitrogenase activities induced by the presence of the AM fungus in lateral roots. The results point to additive effects of interactions between mycorrhizal fungi and rhizobia in increasing the extent of acidification of the “nodulesphere” and the hyposphere.     

Chloroplast phylogeographic patterns of Calligonum sect. Pterococcus (Polygonaceae) in arid Northwest China

To understand the impacts of past climatic change and geological events on the evolutionary history of Calligonum sect. Pterococcus, including C. aphyllum, C. rubicundum and C. leucocladum, a total of 128 individuals from 14 populations, mainly from arid Northwest China, were sampled. Two cpDNA intergenic spacer regions (rpl32‐trnL and ycf6‐psbM) were sequenced and 11 haplotypes were identified. Levels of genetic differentiation between populations was low in C. rubicundum (F_ST = 0.54317, p < 0.001) and C. aphyllum (F_ST = 0.55795), while much higher in C. leucocladum (F_ST = 0.95800, p < 0.001), possibly as an effct of differences in geographic distributions and habitats. Analysis of molecular variance (AMOVA) revealed that most of the total genetic variations occurred among species (72.97%). Among eleven identified haplotypes, only H1 and H2 were shared between C. aphyllum and C. rubicundum, while nine were private for one of the three species. The eleven identified haplotypes were divided into two major clades, but they did not yield three species‐specific lineages. Calligonum sect. Pterococcus therefore not appeared reciprocally monophyletic, more likely due to incomplete lineage sorting than hybridization. Mismatch distribution analysis suggested that only C. aphyllum has experienced recent demographic expansion. Divergence time among the 11 haplotypes was estimated at between 2.84 Ma and 0.06 Ma. Within the two clades, haplotype divergence began in early Pleistocene and mainly occurred during the middle to late Pleistocene and was most likely triggered by Quaternary climatic oscillations and increasing aridity of the region.     

Mass spectrometric U-series dating of Huanglong Cave in Hubei Province,central China: Evidence for early presence of modern humans in eastern Asia

Most researchers believe that anatomically modern humans (AMH) first appeared in Africa 160-190 ka ago, and would not have reached eastern Asia until ∼50 ka ago. However, the credibility of these scenarios might have been compromised by a largely inaccurate and compressed chronological framework previously established for hominin fossils found in China. Recently there has been a growing body of evidence indicating the possible presence of AMH in eastern Asia ca. 100 ka ago or even earlier. Here we report high-precision mass spectrometric U-series dating of intercalated flowstone samples from Huanglong Cave, a recently discovered Late Pleistocene hominin site in northern Hubei Province, central China. Systematic excavations there have led to the in situ discovery of seven hominin teeth and dozens of stone and bone artifacts. The U-series dates on localized thin flowstone formations bracket the hominin specimens between 81 and 101 ka, currently the most narrow time span for all AMH beyond 45 ka in China, if the assignment of the hominin teeth to modern Homo sapiens holds. Alternatively this study provides further evidence for the early presence of an AMH morphology in China, through either independent evolution of local archaic populations or their assimilation with incoming AMH. Along with recent dating results for hominin samples from Homo erectus to AMH, a new extended and continuous timeline for Chinese hominin fossils is taking shape, which warrants a reconstruction of human evolution, especially the origins of modern humans in eastern Asia.     

澳门青洲山翻白叶树群落特征及物种多样性研究

根据样方调查结果,对澳门青洲山翻白叶树(Pterospermum heterophyllum)群落的种类组成、外貌、结构特征与动态及物种多样性进行分析.结果表明:(1)在1 600 m2样地中,有维管束植物47种,隶属于29科44属;对乔木层和灌木层主要物种的重要值以及频度分析结果显示,该群落是以翻白叶树为主的单优种群落,群落种类组成多样性和水平分布不均匀,其外貌终年常绿;对优势种种群的年龄结构分析显示,该种群处于增长状态;(2)整个群落的物种丰富度Margalef指数为13.72,Shannon-Wiener指数为1.15,Simpson指数为0.875,均匀度指数为0.34,该群落的层次格局为:灌木层>藤本层>乔木层>草本层;(3) 与其他5个不同类型地区森林群的物种多样性比较结果显示,澳门翻白叶树群落的物种多样性明显低于其他5个森林群落.     

QCM detection of DNA targets with single-base mutation based on DNA ligase reaction and biocatalyzed deposition amplification

A novel biosensing technique for highly specific identification of gene with single-base mutation is proposed based on the implementation of the DNA ligase reaction and the biocatalyzed deposition of an insoluble product. The target gene mediated deposition of an insoluble precipitate is then transduced by quartz crystal microbalance (QCM) measurements. In this method, the DNA target hybridizes with a capture DNA probe tethered onto the gold electrode and then with a biotinylated allele-specific detection DNA. A ligase reaction is performed to generate the ligation between the capture and the detection probes, provided there is perfect match between the DNA target and the detection probe. Otherwise even when there is an allele mismatch between them, no ligation would take place. After thermal treatment at an elevated temperature, the formed duplex melts apart that merely allows the detection probe perfectly matched with the target to remain on the electrode surface. The presence of the biotinylated allele-matched probe is then detected by the QCM via the binding to streptavidin-peroxide horseradish (SA-HRP), which catalyzes the oxidative precipitation of 3,3-diaminobenzidine (DAB) by H2O2 on the electrode and provides an amplified frequency response. The proposed approach has been successfully implemented for the identification of single-base mutation in -28 site of the beta-thalassemia gene with a detection limit of 0.1 nM, demonstrating that this method provides a highly specific and cost-efficient approach for point mutation detection.     

GC-GAP,a Rho family GTPase-activating protein that interacts with signaling adapters Gab1 and Gab2

Gab1 and Gab2 are scaffolding proteins acting downstream of cell surface receptors and interact with a variety of cytoplasmic signaling proteins such as Grb2, Shp-2, phosphatidylinositol 3-kinase, Shc, and Crk. To identify new binding partners for GAB proteins and better understand their functions, we performed a yeast two-hybrid screening with hGab2-(120-587) as bait. This work led to identification of a novel GTPase-activating protein (GAP) for Rho family GTPases. The GAP domain shows high similarity to the recently cloned CdGAP and displays activity toward RhoA, Rac1, and Cdc42 in vitro. The protein was named GC-GAP for its ability to interact with GAB proteins and its activity toward Rac and Cdc42. GC-GAP is predominantly expressed in the brain with low levels detected in other tissues. Antibodies directed against GC-GAP recognized a protein of approximately 200 kDa. Expression of GC-GAP in 293T cells led to a reduction in active Rac1 and Cdc42 levels but not RhoA. Suppression of GC-GAP expression by siRNA inhibited proliferation of C6 astroglioma cells. In addition, GC-GAP contains several classic proline-rich motifs, and it interacts with the first SH3 domain of Crk and full-length Nck in vitro. We propose that Gab1 and Gab2 in cooperation with other adapter molecules might regulate the cellular localization of GC-GAP under specific stimuli, acting to regulate precisely Rac and Cdc42 activities. Given that GC-GAP is specifically expressed in the nervous system and that it is localized to the dendritic processes of cultured neurons, GC-GAP may play a role in dendritic morphogenesis and also possibly in neural/glial cell proliferation.