Identification of antiviral-relevant genes in the cultured fish cells induced by UV-inactivated virus

UV-inactivated grass carp hemorrhage virus (GCHV) can induce high titer of interferon in cultured CAB (Crucian carp (Carassius auratus L.) blastulae )cells,and thus defend host cells against the virus invasion ,The mechanism is proposed that an antiviral state should be established in the host cells by activating expression of a set of antiviral-relevant genes,In this study ,suppressive subtractive hybridization is applied to constructing a subtracted ,cDNA library with mRNAs isolated from UV-inactivated GCHV infected and mock-infected CAB cells,272 differential cDNA fragments are identified by both PCR and dot blot from the subtractive cDNA library .Sequencing analysis reveals 69 genes,including 46 known gene homlologues,and 23 unknown putative genes,The known genes include the gemes involved in interferon signaling pathways,such as Stat1 and Jak1,the antiviral gences,such as Mx and Vipering,and a set of interferon-stimulated genes observed in mammalian cells. Most of the unknow putative genes contain AU-rich element in their sequences,Differential expressions of these genes are further confirmed by virtual Northern blot and RT-PCR,The data imply that UV-inactivated GCHV is not only able to induce production of interferon in the infected CAB cells,but also leads to the expression of a series of antiviral-relevant genes or immune-releveant genes,and therefore reveals that the signaling pathway of interferon system and antiviral mechanism in fish are similar to those in mammals.     

The molecular mechanism of embryonic stem cell pluripotency maintenance

In vitro cultured embryonic stem （ES） cells are derived from the inner cell mass （ICM） of pre-implantation embryos, and are capable of giving rise to all cell and tissue types of the three germ layers upon being injected back into blastocysts. These ceils are therefore said to possess pluripotency that can be maintained infinitely in culture under optimal conditions. Such pluripotency maintenance is believed to be due to the symmetrical cleavage of the cells in an undifferentiated state. The pluripotency of ES cells is the basis for their various practical and potential applications. ES cells can be used as donor cells to generate knockout or transgenic animals, as in vitro models of mammalian development, and as cell resources for cell therapy in regenerative medicine. The further success in these applications, particularly in the last two, is dependent on the establishment of a culture system with components in the medium clearly defined and the subsequent procedures for controlled differentiation of the cells into specific lineages. In turn, elucidating the molecular mechanism for pluripotency maintenance of ES cells is the prerequisite. This paper summarizes the recent progresses in this area, focusing mainly on the LIF/STAT3, BMPs/Smads, canonical Wnt, TGFβ/activin/nodal, PI3K and FGF signaling pathways and the genes such as oct4, nanog that are crucial in ES cell pluripotency maintenance. The regulatory systems of pluripotency maintenance in both mouse and human ES cells are also discussed. We believe that the cross-talkings between these signaling pathways, as well as the regulatory system underlying pluripotency maintenance will be the main focus in the area of ES cell researches in the future.     

Identification of differentially expressed genes in esophageal cancer through SSH in combination with high throughput reverse Northern screening

To understand the molecular mechanisms of carcinogenesis of esophagus and to isolate genes with different expression levels in esophageal cancer, suppression subtractive hybridization (SSH) was combined with PCR-based cDNA synthesis and reverse Northern on the cancer tissues and matched almost normal mucosa using 5 microgram of total RNA as starting marterial. Eight genes were found expressed differentially in esophageal cancer, in which 5 were known genes and 3 were novel ones; and 6 were down-regulated in cancer tissues, while 2 were up-regulated; 6 were of mid-high abundance and 2 were of low abundance in esophagus. The results revealed that alteration in expression level of multiple genes underlied the initiation and development of esophageal cancer. The differentially expressed genes identified in this study such as liporcotinⅠ, cystatin A, cystatin B, cytokeratin 13 may play roles in dedifferentiation, transformation and malignant proliferation of esophageal cancer. The combination of SSH with PCR-based double- strand cDNA synthesis and high throughput reverse Northern screening is an efficient way to isolate differentially expressed genes from microgram of total RNA.     

Maturation of Osteoblast-Like Saos-2 on Hydroxyapatite Ceramics

Hydroxyapatite ceramics (.HA) has been proved to be excellent in biocompatibility and bioactivity.However, limited information is available concerning how HA ceramics affects the maturation of es-tcoblasts in molecular biological level /n v/tro. This study examines the mRNA expression and protein production of hone-related genes in estcoblast-like cell line(Saos-2) cultured on HA disks. Saos-2 cells are seeded onto the substrates and cultured for 18 days. Harvested cells are tested for the cell growth rate, expression of mRNAs for esteocalcin and alkaline phosphatase, and protein production of bone sialo-protein and esteocalcin. MTS assay shows that cell proliferates well on HA ceramic substrate. After 9d,bone sialoprotein and esteocalcin protein production in SAPS-2 increases more on HA surfaces than on control material. As bone sialoprotein and esteocalcin are the genes to be highly expressed at the late stage of estcoblast differentiation, this study reveals that after long time culture in HA, HA can induce Saos-2 maturation. The behavior of Saos-2 on HA surfaces revealed in this study provides valuable infor-mation for the understanding of the biocompatibility and bioactivity of HA ceramics.     

Metformin targets liver tumor-initiating cells through the PI3K/Akt/mTOR survival pathway

Liver tumor-initiating cells （T-ICs） are thought to be inherently resistant to the cytotoxic effects of chemo- therapy, and can self-renewal and maintain tumor-initiating potential. Therefore, effective anticancer research strategies should target the unique properties of T-ICs. In this study, we found that metformin, a first-line drug of choice for the treatment of type 2 diabetes, inhibited liver T-ICs both in vivo and in vitro. Metformin inhibited the formation of hepato- spheres and epithelial-specific antigen-positive （ESA, CD133＋） cell colonies by hepatocellular carcinoma （HCC） cell lines. Metformin also downregulated the expression of several T-IC-related genes which are involved in the signal- ing pathways, governing the self-renewal, proliferation and differentiation of T-ICs. Furthermore, the targeting of liver T-ICs by metformin was PI-3-kinase-Akt-mTOR （PI3K/Akt/ mTOR）-pathway dependent. The PI3K/Akt/mTOR inhibitorLY294002 and rapamycin abolished the inhibitory effect of metformin on CD133＋ cells, and the PI3K/Akt/mTOR stimulator EGF promoted the inhibitory effect of mefformin on CD 133＋ cells. Metformin also dramatically decreased the tumor volume and number of CD133 expressing tumor cells in a xenograft mouse model. Mefformin exerted a synergistic effect with cisplatin to target both T-ICs and non-T-ICs, and resulted in the smallest tumor volume and lowest number of CD133 expressing tumor cells. This study indicates that the antidiabetic drug metformin could potentially be used in combination therapy with chemotherapeutic agents to improve the treatment of liver cancer.     

Myofibrillogenesis regulator-1 promotes cell adhesion and migration in human hepatoma cells

Myofibrillogenesis regulator-1 (MR-1) is a gene overexpressed usually in many human cancers. However, the effects of MR-1 on cell proliferation, adhesion, migration and genome-wide gene regulation are still unclear. In this study, a human hepatoma cell line that highly overexpresses MR-1, BEL-7402/MR-1 cells was established. While the high expression of MR-1 did not promote cell proliferation, it significantly increased cell spreading, adhesion and migration compared with control cells. A total of 147 genes were regulated by MR-1 expression, 46 genes were down-regulated and 101 genes were up-regulated by MR-1 overexpression. Many of these genes were related to cell adhesion, cytoskeletal regulation, MAPK signaling, and cell cycle related pathways. Western blot analysis further confirmed the regulation of pathways associated with migration by MR-1. These results suggest that MR-1 is involved in the regulation of cancer cell adhesion, migration and related gene expression.     

木豆叶水提物对兔破骨细胞样细胞的形成及其骨吸收功能的影响

通过对骨髓单核细胞诱导分化形成的抗酒石酸酸性磷酸酯酶(TRAP)阳性多核巨细胞计数, 研究了木豆叶水提物对1,25-二羟基维生素D3诱导兔骨髓单核细胞分化形成破骨细胞样细胞的影响.通过破骨细胞样细胞与骨片共培养的方法,用显微摄影结合计算机图像分析测定骨吸收造成的陷窝数目及表面积,研究了其对破骨细胞样细胞骨吸收功能的影响.结果表明, 在0.01～100 mg/L质量浓度内, 木豆叶水提物能够剂量依赖性地抑制破骨细胞样细胞的形成(P<0.05),同时也明显抑制破骨细胞样细胞的骨吸收活性(P<0.05),具体表现在, 与对照组比较,其骨吸收陷窝数目和表面积明显减少,但没有呈现出质量浓度依赖性, 木豆叶水提物对破骨细胞样细胞骨吸收指数的影响的结果给予了进一步的证实. 这提示木豆叶水提物对骨质疏松症患者可能有一定的疗效.     

Analysis of gene expression patterns with cDNA microarray during late stage of spermatogenesis in mice

The differentiation process of round spermatids to spermatozoa during the late stage of spermatogenesis is called spermiogenesis. To explore spermiogenesis-related genes, cDNA microarray was used to study expression patterns of 1176 genes in pachytene spermatocytes, round spermatids and elongating spermatids of Balb/c mice. The results showed that 208 genes were detected in all the three cell types. Most of them were down-regulated from pachytene spermatocytes to round spermatids and elongating spermatids. However, up-regulation of 7 genes expression in round spermatids and 3 genes in elongating spermatids were found. Expression of 7 differentially expressed genes in cDNA arrays was further confirmed by semi-quantitative RT-PCR study. The RT-PCR results indicated that the expression of 6 genes was consistent with that in cDNA arrays, only one gene did not show differential expression by RT-PCR. These results may provide important clues for studying of expression, regulation, and function of spermiogenesis-related genes.     

Genomic analysis of metastasis reveals an essential role for RhoC

The most damaging change during cancer progression is the switch from a locally growing tumour to a metastatic killer. This switch is believed to involve numerous alterations that allow tumour cells to complete the complex series of events needed for metastasis. Relatively few genes have been implicated in these events. Here we use an in vivo selection scheme to select highly metastatic melanoma cells. By analysing these cells on DNA arrays, we define a pattern of gene expression that correlates with progression to a metastatic phenotype. In particular, we show enhanced expression of several genes involved in extracellular matrix assembly and of a second set of genes that regulate, either directly or indirectly, the actin-based cytoskeleton. One of these, the small GTPase RhoC, enhances metastasis when overexpressed, whereas a dominant-negative Rho inhibits metastasis. Analysis of the phenotype of cells expressing dominant-negative Rho or RhoC indicates that RhoC is important in tumour cell invasion. The genomic approach allows us to identify families of genes involved in a process, not just single genes, and can indicate which molecular and cellular events might be important in complex biological processes such as metastasis.     

Identification of antiviral- relevant genes in the cultured fish cells induced by UV-inactivated virus

~~Identification of antiviral- relevant genes in the cultured fish cells induced by UV-inactivated virus~~     

三维培养体系下EGCG对乳腺癌干细胞迁移性和自我更新的抑制效应

以乳腺癌细胞三维培养体系为基础,探究(-)-表没食子儿茶素-3-没食子酸酯((-)-Epigallocatechin-3-gallate,EGCG)对乳腺癌干细胞(BCSCs)的药理学作用.细胞迁移性实验结果发现,20和80μmol/L EGCG能显著抑制迁移性基因的转录水平,降低通过Transwell穿膜细胞的数量,延缓癌细胞划痕修复时间,抑制癌细胞迁移性.在对BCSCs自我更新研究中,EGCG通过下调干细胞关键基因的转录而抑制癌细胞单克隆形成和克隆形成能力,抑制癌细胞自我更新,并有效降低CD24-亚群BCSCs含量.研究结果表明,在三维培养条件下,EGCG对BCSCs的迁移性和自我更新具备显著的抑制效应.     

大鼠再生肝8种细胞的嘌呤核苷酸代谢基因转录谱预示的代谢活动

为了解大鼠肝再生中肝细胞、胆管上皮细胞、卵圆细胞、星形细胞、窦内皮细胞、库普弗细胞、陷窝细胞、树突状细胞等8种肝脏细胞的嘌呤核苷酸代谢基因转录谱及预示的代谢活动,按张丽君[1]等方法分离大鼠部分肝切除后10个恢复时间点大鼠再生肝的上述8种细胞,用RatGenome2302.0芯片等检测嘌呤核苷酸代谢基因在上述细胞中表达变化,用Excel等软件及生物信息学和系统生物学等方法分析它们的表达模式、预示的生理活动等.结果表明,85个嘌呤核苷酸代谢基因在大鼠肝再生中发生了有意义表达变化,8种细胞的相应基因数为40,43,30,42,22,26,36和48.上调、下调、上/下调的基因个数为49、11、31,相应细胞的基因数为27、20和1,31、4和1,15、7和3,12、10和0,23、15和3,19、7和2,39、3和1,33、6和0.其中,催化DNA合成的DNA聚合酶基因和催化RNA合成的RNA聚合酶基因在肝再生的多个时间点和多种细胞中表达增强,胆管上皮细胞和星形细胞的腺苷酸合成相关基因表达增强.肝细胞、卵圆细胞和星形细胞的核苷酸分解相关基因、肝细胞、星形细胞和树突状细胞的嘌呤核苷分解相关基因表达减弱.预示大鼠肝再生与嘌呤核苷酸代谢密切相关.     

Control of neuronal fate by the Drosophila segmentation gene even-skipped

The central nervous system (CNS) contains a remarkable diversity of cell types. The molecular basis for generating this neuronal diversity is poorly understood. Much is known, however, about the regulatory genes which control segmentation and segment identity during early Drosophila embryogenesis. Interestingly, most of the segmentation and homoeotic genes in Drosophila, as well as many of their vertebrate homologues, are expressed during the development of the nervous system (for example, ref. 3). Are these genes involved in specifying the identity of individual neurons during neurogenesis, just as they specify the identity of cells during segmentation? We previously described the CNS expression of the segmentation gene fushi tarazu (ftz) and showed that ftz CNS expression is involved in the determination of an identified neuron. Here we show that another segmentation gene, even-skipped (eve), is expressed in a different but overlapping subset of neurons. Temperature-sensitive inactivation of the eve protein during neurogenesis alters the fate of two of these neurons. Our results indicate that the nuclear protein products of the eve and ftz segmentation genes are components of the mechanism controlling cell fate during neuronal development.     

Cloning and analysis of a cDNA encoding acetohydroxy acid isomeroreductase from G2 pea

Using cDNA representational difference analysis (cDNA RDA) method, we have successfully isolated a gene fragment whose expression was specifically induced by external GA₃ application. Screening a G2 pea cDNA library using this fragment as a probe, we obtained a 2036 bp full-length cDNA. It contains a 1746 bp open reading frame and encodes a protein of 581 amino acids with a theoretical molecular weight of 64 ku. It shares high-level sequence identity withAAIR genes from other plant species. This cDNA was cloned into expression vector and recombinantE. coli DH5α cells with remarkable AAIR enzyme activity were obtained.