Reduction of Orc6 Expression Sensitizes Human Colon Cancer Cells to 5-Fluorouracil and Cisplatin

Previous studies from our group have shown that the expression levels of Orc6 were highly elevated in colorectal cancer patient specimens and the induction of Orc6 was associated with 5-fluorouracil (5-FU) treatment. The goal of this study was to investigate the molecular and cellular impact of Orc6 in colon cancer. In this study, we use HCT116 (wt-p53) and HCT116 (null-p53) colon cancer cell lines as a model system to investigate the impact of Orc6 on cell proliferation, chemosensitivity and pathways involved with Orc6. We demonstrated that the down regulation of Orc6 sensitizes colon cancer cells to both 5-FU and cisplatin (cis-pt) treatment. Decreased Orc6 expression in HCT-116 (wt-p53) cells by RNA interference triggered cell cycle arrest at G1 phase. Prolonged inhibition of Orc6 expression resulted in multinucleated cells in HCT-116 (wt-p53) cell line. Western immunoblot analysis showed that down regulation of Orc6 induced p21 expression in HCT-116 (wt-p53) cells. The induction of p21 was mediated by increased level of phosphorylated p53 at ser-15. By contrast, there is no elevated expression of p21 in HCT-116 (null-p53) cells. Orc6 down regulation also increased the expression of DNA damaging repair protein GADD45β and reduced the expression level of JNK1. Orc6 may be a potential novel target for future anti cancer therapeutic development in colon cancer.     

金佛拟小鲵幼体皮肤的显微结构观察

采用光镜和扫描电镜对金佛拟小鲵(Pseudohynobius jinfo)幼体皮肤进行组织学和形态学观察。金佛拟小鲵幼体皮肤由表皮和真皮构成。不同部位皮肤厚度不同,头部背侧皮肤最薄,其厚度为(45.99±12.77)μm,尾部腹侧的皮肤最厚,其厚度为(95.21±42.72)μm。表皮角质层仅躯干背部和尾部明显,由仍具有一定生理活性的复层扁平上皮细胞构成。皮肤腺体包括黏液腺和颗粒腺。黏液腺广泛分布于身体各个部位的皮肤,颗粒腺呈区域性分布,仅见躯干部和尾部皮肤,其体积大于黏液腺。毛细血管多分布于真皮疏松层腺体周围,与表皮层紧密接触并凸向表皮。色素细胞主要分布于表皮和疏松层的交界处,呈多细胞聚集的状态,形成厚度不一的色素层。     

Comparative Genomic Analysis of N2-Fixing and Non-N2-Fixing Paenibacillus spp.: Organization,Evolution and Expression of the Nitrogen Fixation Genes

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N₂-fixing strains. The heterogeneity of the 31 genomes (15 N₂-fixing and 16 non-N₂-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N₂-fixing strains. The nif cluster is under control of a σ⁷⁰-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe–S] cluster is highly conserved in N₂-fixing and non-N₂-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N₂-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N₂-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N₂-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation.     

Determination of zeranol and its metabolites in bovine muscle and liver by a chemiluminescence enzyme immunoassay: compared to an ultraperformance liquid chromatography tandem mass spectroscopy method

A chemiluminescent enzyme immunoassay (CLEIA) was compared to an ultraperformance liquid chromatography tandem mass spectroscopy (UPLC‐MS/MS) procedure for the analysis of zeranol and its metabolites in bovine tissue samples. Apparent recoveries from fortified samples by both methods were comparable at 0.5–4.0 µg/kg and a significant correlation was obtained. For CLEIA analysis, hapten mimicking the analyte was first synthesized and conjugated with the carrier protein bovine serum albumin as the immunogen to produce monoclonal antibody. The obtained antibody showed extensive cross‐reactivity toward zeranol metabolites (zearalanone). The limit of detection of CLEIA and UPLC‐MS/MS was 0.05 µg/kg and 0.5 µg/kg, respectively. Recoveries of both methods for fortified samples were higher than 75.0% with the coefficient of variation less than 15%. These results indicated that the combination of screening with CLEIA and confirmation with UPLC‐MS/MS for zeranol and its metabolites would be a reliable method for a large number of bovine samples. Copyright © 2013 John Wiley & Sons, Ltd.     

桑蚕金属硫蛋白基因在大肠杆菌中的克隆和表达

用\%Bam\%HⅠ和\%Sac\%Ⅰ双酶切质粒pCM1\|1,获得酵母的MTI基因片段,用非放射性地高辛标记作为探针。提取桑蚕肥苏蚕卵的总DNA,分别用\%Eco\%RⅠ、\%Bam\%HⅠ和\%Hin\%dⅢ酶切,与MTI探针进行Southern杂交,出现较强的杂交信号。然后用\%Eco\%RⅠ完全酶切桑吞的总DNA,电洗脱法回收1～6kb的染色体片断,与\%Eco\%RⅠ酶切的M13-载体以3∶1比例连接,转化受体菌DH5α。筛选到4 000多个白色转化子,与探针MTI进行Southern杂交筛选阳性转化子,选择到有强杂交信号的三个转化子［编号为T1(pZHC\|1)\,T5(pZHC\|5)\,T7(pZHC\|7)\]。用12种限制性内切酶对pZHC\|5重组质粒进行酶切分析表明插入片段约12kb,在基因内有一个\%Hin\%dⅢ位点。抗性测定表明受体菌DH5α在含有50mmol/L CuSO\-4的培养基上生长,在含有52mmol/L CuSO\-4的培养基上不生长,而转化子确能在含有52mmol/L CuSO\-4以上的培养基上生长。上述研究结果表明12kb左右的插入片段含有桑吞的金属硫蛋白基因。     

A chemical-genetic approach to studying neurotrophin signaling

Trk tyrosine kinases are receptors for members of the neurotrophin family and are crucial for growth and survival of specific populations of neurons. Yet, the functions of neurotrophin-Trk signaling in postnatal development as well as maintenance and plasticity of the adult nervous system are less clear. We report here the generation of mice harboring Trk knockin alleles that allow for pharmacological control of Trk kinase activity. Nanomolar concentrations of either 1NMPP1 or 1NaPP1, derivatives of the general kinase inhibitor PP1, inhibit NGF and BDNF signaling in TrkA(F592A) and TrkB(F616A) neurons, respectively, while no such Trk inhibition is observed in wild-type neurons. Moreover, oral administration of 1NMPP1 leads to specific inhibition of TrkA(F592A), TrkB(F616A), and TrkC(F167A) signaling in vivo. Thus, Trk knockin mice provide valuable tools for selective, rapid, and reversible inhibition of neurotrophin signaling in vitro and in vivo.     

CD8+ DC,but Not CD8?DC,Isolated from BCG-Infected Mice Reduces Pathological Reactions Induced by Mycobacterial Challenge Infection

Background

Tuberculosis is a mycobacterial infection causing worldwide public health problems but the available vaccine is far from ideal. Type-1 T cell immunity has been shown to be critical for host defence against tuberculosis infection, but the role of dendritic cell (DC) subsets in pathogenesis of mycobacterial infection remains unclear.

Methodology/Principal Findings

We examined the effectiveness of dendritic cell (DC) subsets in BCG-infected mice in generating immune responses beneficial for pathogen clearance and reduction of pathological reactions in the tissues following challenge infection. Our data showed that only the adoptive transfer of the subset of CD8α⁺ DC isolated from infected mice (iCD8⁺ DC) generated significant protection, demonstrated by less mycobacterial growth and pathological changes in the lung and liver tissues in iCD8⁺ DC recipients than sham-treated control mice. The adoptive transfer of the CD8α⁻DC from the infected mice (iCD8⁻ DC) not only failed to reduce bacterial growth, but enhanced inflammation characterized by diffuse heavy cellular infiltration. Notably, iCD8⁻ DC produced significantly higher levels of IL-10 than iCD8⁺ DC and promoted more Th2 cytokine responses in in vitro DC-T cell co-culture and in vivo adoptive transfer experiments.

Conclusions/Significance

The data indicate that in vivo BCG-primed CD8⁺ DC is the dominant DC subset in inducing protective immunity especially for reducing pathological reactions in infected tissues. The finding has implications for the rational improvement of the prophylactic and therapeutic approaches for controlling tuberculosis infection and related diseases.     

Alterations of adenylyl cyclase and G proteins in aortocaval shunt-induced heart failure

Unlike most other experimental models of congestive heart failure, the volume overload model induced by aortocaval shunt (AVS) in rats was found to exhibit enhanced beta-adrenoceptor (beta-AR) signaling. To study whether the adenylyl cyclase (AC)-G protein system is involved in such a change, we examined cardiac AC activity and protein content as well as G(s)alpha and G(i)alpha activities, protein contents, and mRNA levels in both left (LV) and right (RV) ventricles at the failing stage (16 wk after surgery). Basal and forskolin-stimulated AC activities were significantly increased in both LV and RV from the failing hearts; this change was associated with an upregulation of type V/VI AC protein. In contrast to 5'-guanylyl imidodiphosphate and NaF, the stimulatory effect of isoproterenol on AC was increased in the failing heart. Although G(s)alpha and G(i)alpha protein contents in the failing hearts were not altered, the mRNA level for G(s)alpha was decreased by 20% and that for G(i)alpha was increased by 20%. In addition, the activity of G(s)alpha, but not G(i)alpha, as assessed by toxin-catalyzed ADP ribosylation, was significantly decreased in the failing heart. Losartan and imidapril treatments improved cardiac function and attenuated alterations in mRNA levels for G(s)alpha and G(i)alpha proteins, as well as G(s)alpha activity, without affecting changes in AC protein content or activities in heart failure due to volume overload. These data suggest that increased AC activity may contribute to the enhanced beta-AR signaling in the AVS model of heart failure, whereas alterations in gene expression for G proteins may be of an adaptive nature at this stage of heart failure.     

Adsorption of DNA by Bacteria and Their Composites with Minerals

Previous studies revealed the thermodynamic properties of DNA adsorption on pure minerals or biomasses; however, there has been little attempt to develop such studies on bacteria–mineral composites. Equilibrium adsorption experiments, attenuated total reflectance Fourier transform infrared spectroscopy, and isothermal titration calorimetry were employed to investigate the adsorption of DNA by Bacillus subtilis, Pseudomonas putida, and their composites with minerals. Similar capacity and affinity were observed for DNA adsorption on two bacterial cells. However, different patterns were found in the adsorption of DNA by bacteria–mineral composites. The Gram-positive bacterium B. subtilis enhanced the adsorption of DNA on its mineral composites compared with their individual components, while the composites of Gram-negative bacterial cells with kaolinite and goethite bound lower amounts of DNA than the predicted values. The thermodynamic parameters and the Fourier transform infrared spectra showed that van der Waals force and hydrogen bonding are responsible for the DNA adsorption on B. subtilis–minerals and P. putida–kaolinite. By contrast, the entropy increases of excluded water rearrangement and dehydration effect play key roles in the interaction between DNA and P. putida–montmorillonite/goethite composites.     

Species diversity and interspecific association in development sequence of Hippophae rhamnoides plantations in the Loess Hilly Region, China

Based on our field investigation, we analyzed the characteristics of species diversity and interspecific association at different development stages of Hippohae rhamnoides plantations. The results show that the species diversities of the shrub layer, grass layer and the whole community of the H. rhamnoides plantations all fluctu-ated in an "S" shape pattern. At different development stages, the species richness and diversity were higher in the grass layer than in the shrub layer. The shrub species diversity was lower on bare land, but increased gradually with development of the community. The shrub evenness index was higher in 13-year forest stand, while the grass diversity index was higher in the 3-year and 25-year plan-tations and lower in the 8-year plantation. The positive and negative absolute values of interspecific association between H. rhamonides and other principal species chan-ged in a parabolic pattern. The significance level and intensity of interspecific association were lower or weaker in thel3-year plantation, indicating that the species sub-stitution rate slowed down, competition became less intense and community composition and its structure reached a relatively stable state. Therefore, to improve the ecological environment, priority should be given to the protection of the H. rhamnoides plantations younger than 13 years, while for those of 25 year plantations, mod-erate thinning should be made to promote their regenera-tion.