A “GC-rich” method for mammalian gene expression: A dominant role of non-coding DNA GC content in regulation of mammalian gene expression

High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein. The stable expression vectors were constructed by inserting a naturally-occurring 1.006 kb or a synthetic 0.733 kb DNA fragment (including intron) of extremely GC-rich at the 5′ or/and 3′ flanking regions of these protein genes or their gene promoters. This experiment is the first experimental evidence showing that a non-coding extremely GC-rich DNA fragment is a super “chromatin opening element” and plays an important role in mammalian gene expression. This experiment has further indicated that chromatin-based regulation of mammalian gene expression is at least partially embedded in DNA primary structure, namely DNA GC-content.     

Engineering a homobutanol fermentation pathway in Escherichia coli EG03

A homobutanol fermentation pathway was engineered in a derivative of Escherichia coli B (glucose [glycolysis] => 2 pyruvate + 2 NADH; pyruvate [pyruvate dehydrogenase] => acetyl-CoA + NADH; 2 acetyl-CoA [butanol pathway enzymes] + 4 NADH => butanol; summary stoichiometry: glucose => butanol). Initially, the native fermentation pathways were eliminated from E. coli B by deleting the genes encoding for lactate dehydrogenase (ldhA), acetate kinase (ackA), fumarate reductase (frdABCD), pyruvate formate lyase (pflB), and alcohol dehydrogenase (adhE), and the pyruvate dehydrogenase complex (aceEF-lpd) was anaerobically expressed through promoter replacement. The resulting strain, E. coli EG03 (ΔfrdABCD ΔldhA ΔackA ΔpflB Δ adhE ΔpdhR ::pflBp6-aceEF-lpd ΔmgsA), could generate 4 NADH for every glucose oxidized to two acetyl-CoA through glycolysis and the pyruvate dehydrogenase complex. However, EG03 lost its ability for anaerobic growth due to the lack of NADH oxidation pathways. When the butanol pathway genes that encode for acetyl-CoA acetyltransferase (thiL), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd, etfA, etfB), and butyraldehyde dehydrogenase (adheII) were cloned from Clostridium acetobutylicum ATCC 824, and expressed in E. coli EG03, a balanced NADH oxidation pathway was established for homobutanol fermentation (glucose => 4 NADH + 2 acetyl-CoA => butanol). This strain was able to convert glucose to butanol (1,254 mg l(-1)) under anaerobic condition.     

激素对转基因雪莲毛状根植株再生及类黄酮产生的影响

为了研究外源激素GA3和IAA对3个转基因新疆雪莲类黄酮高产毛状根系C17、C27、C46的植株再生及其总黄酮含量的影响,在培养基中添加不同浓度的GA3和IAA,结果发现,GA3浓度高于1.0 mg/L时,可诱导毛状根系产生不定芽,其中以GA3浓度为2.0 mg/L时,转基因毛状根系C17的不定芽再生率最高,可达82％。高压液相色谱以及紫外分光光度法测定结果表明,与未用激素处理的毛状根和它的再生植株相比,外源激素GA3和IAA能显著提高毛状根培养物中芹菜素和总黄酮的含量。毛状根系的组织干重与类黄酮的含量没有相关性,但毛状根系的再生率与类黄酮的含量几乎呈反相关性。     

AP-3 Mediates Tyrosinase but Not TRP-1 Trafficking in Human Melanocytes

Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the beta 3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the beta 3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3-deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.     

The inhibitory effect of BSP-A1/-A2 on protein kinase C and tyrosine protein kinase

Bovine seminal plasma contains a group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), and they are secreted by the seminal vesicles. In our study, we purified the BSP-A1/-A2 through affinity chromatography and found for the first time that BSP-A1/-A2 can inhibit the activity of protein kinase C (PKC) and tyrosine protein kinase (TPK). The inhibition was dose dependent. When the PKC and TPK activities are expressed as the logarithm of percentage activity taking the activity in the absence of the BSP-A1/-A2 as 100%, there is a linear relationship between the their activities and the dose of BSP-A1/-A2.     

顶青霉D_(15)木聚糖酶性质研究

本文对项青霉D_(1(?))的四个木聚糖酶组分的特性进行了研究。木聚糖酶组分D_(x1)、D_(x4)的最佳反应pH为4.8,最适温度分别为40℃和50℃,D_(x2)和D_(x3)的最适pH和温度都分别为pH4.2和50℃。Ag~(++)、Hg~(++),Cu~(++)对四个组分的活性均有强烈的抑制作用,SDS也能产生明显的抑制效果。Mn~(++)对D_(x1)具有促进作用。D_(x1)、D_(x4)在以燕麦木聚糖为底物时活性最高,其Km值分别为11.7(mg/ml)和8.3(mg/ml),D_(x2)和D_(x3)则分别在水解红麻杆木聚糖和落叶松木聚糖时活性最强,Km值分别为8.4(mg/ml)和6.3(mg/ml)。水解燕麦木聚糖,D_(x1)的产物主要为木糖,同时带有少量的低聚木糖。D_(x2)、D_(x3)和D_(x4)的产物则包括木糖和较多的低聚木糖。D_(x4)与D_(x2)及D_(x3)之间在水解燕麦木聚糖时存在协同作用关系。     

人胸腺上皮细胞培养及其分泌产物

本文通过降低培养基中血清含量,向RPMI 1640培养基中补加三碘甲腺原氨酸而获得一种人胸腺网状上皮细胞占优势生长的培养物。在此培养基中细胞经传代培养长达90天,仍维持正常形态特征。胸腺组织在培养14天后,新生细胞的突起形成网状结构,细胞化学检查和电镜观察表明具有丰富的分泌颗粒,囊泡及张力原纤维束和桥粒等上皮细胞特征。收集合并细胞培养液,经部分纯化后检查其生物活性,表现出具有促进玫瑰花结形成和降低胸腺细胞TdT活性的作用,说明培养细胞的分泌产物具有胸腺激素活性。根据形态学,细胞化学和生物活性检测结果,我们倾向于认为该培养物主要为网状上皮细胞。     

Involvement of Annexin A2 in p53 induced apoptosis in lung cancer

Tumor suppressor p53 plays important roles in cell cycle regulation, apoptosis and DNA repair in different cell types including lung cancer. There are different p53 apoptotic pathways in high and low metastatic ability lung cancer cells. However, the exactly mechanism in the pathway is still unclear. Here we found that Annexin A2, a Ca²⁺-dependent phospholipid-binding protein, is involved in p53-mediated apoptosis. First, by using mRNA differential display technique, down-regulated Annexin A2 expression was found in all cell lines transfected of Ad-p53 (adenoviral expression construct encoding wild type p53 gene) especially in highly metastatic Anip973 lung cancer cells. Then, decreased expression of Annexin A2 was further confirmed by Northern blot and Western blot analysis. At last, knock down of Annexin A2 by siRNA inhibited cellular proliferation in BE1 cell line with highly metastatic ability. Taken together, our results suggested that Annexin A2 may play roles in p53 induced apoptosis and it is also involved in regulation of cell proliferation. The authors Yun Huang, Yan Jin and Cheng-hui Yan contributed equally to this work.     

Choline Transporters in Human Lung Adenocarcinoma： Expression and Functional Implications

Choline is an essential nutrient for cell survival and proliferation, however, the expression and function of choline transporters have not been well identified in cancer. In this study, we detected the mRNA and protein expression of organic cation transporter OCT3, carnitine/cation transporters OCTN 1 and OCTN2, and choline transporter-like protein CTL1 in human lung adenocarcinoma cell lines A549, H 1299 and SPC-A-1. Their expression pattern was further confirmed in 25 human primary adenocarcinoma tissues. The choline uptake in these cell lines was significantly blocked by CTL1 inhibitor, but only partially inhibited by OCT or OCTN inhibitors. The efficacy of these inhibitors on cell proliferation is closely correlated with their abilities to block choline transport. Under the native expression of these transporters, the total choline uptake was notably blocked by specific PI3K/AKT inhibitors. These results describe the expression of choline transporters and their relevant function in cell proliferation of human lung adenocarcinoma, thus providing a potential ＂choline-starvation＂ strategy of cancer interference through targeting choline transporters, especially CTL1.     

鱼类多样性监测的理论方法及中国内陆 水体鱼类多样性监测

近年来, 生物多样性监测网络的建设得到广泛重视, 全球、地区或国家生物多样性观测网不断组建。生物多样性观测的理论框架得到发展, 提出了生物多样性核心监测指标(Essential Biodiversity Variables, EBV)。鱼类多样性监测的理论框架包含于生物多样性核心监测指标之内, 在遗传、物种、生态系统等多层次进行。基于鱼类监测提出的生物完整性指数(index of biotic integrity, IBI)强调不同物种的生态功能, 可以综合反映群落结构和功能的变化, 得到广泛应用。鱼类多样性的监测方法是传统网具和现代水声学等方法的结合。监测结果的分析可以进行简单的指数比较, 也可以进行长期的趋势分析, 寻找关键节点, 探讨宏观生态格局的变化。中国内陆水体鱼类多样性监测网隶属于中国生物多样性监测与研究网络, 拟选取长江、黄河、黑龙江、珠江、澜沧江、怒江、塔里木河及青海湖8大流域, 对25个重要区域和24个重点物种(类群)进行监测, 从重要区域鱼类群落结构、重点物种(类群)种群动态和个体生物学特征、遗传多样性、早期资源等不同层次, 全面监测我国内陆水体鱼类生物多样性状况。