Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.     

体外重构STUB1介导α-1抗胰蛋白酶突变体Z蛋白泛素化修饰反应体系

α-1抗胰蛋白酶Z型突变体蛋白(α-1 antitrypsin Z-mutant protein, ATZ)是引发α-1抗胰蛋白酶缺陷症(α-1 antitrypsin deficiency, AATD)的主要原因,研究ATZ蛋白的泛素化修饰和降解对于治疗AATD具有重要意义。STUB1是一种重要的E3泛素连接酶,参与调节多种蛋白质的泛素化修饰。然而,STUB1是否参与ATZ的泛素化修饰尚未明确。本研究首先将ATZ和STUB1的编码基因克隆到pET28a质粒,构建了这2个蛋白的表达质粒。随后,将重组质粒转入大肠杆菌表达系统,在优化诱导条件实现了重组蛋白的异源表达。通过金属螯合亲和层析技术纯化得到目的蛋白,并通过蛋白质谱分析验证了其氨基酸序列的准确性。利用纯化的ATZ和STUB1重组蛋白,构建了一个体外泛素化修饰反应体系。实验结果显示,在ATP、E1泛素激活酶和E2泛素结合酶的协同作用下,STUB1成功催化了ATZ的泛素化修饰。本研究提供了一种体外获得Z型突变体ATZ纯化蛋白的方法,并确认了STUB1介导ATZ的泛素化修饰功能,推进了对α-1抗胰蛋白酶Z型突变体蛋白在细胞内降解过程的调控机制的理解。     

Design,synthesis, and biological evaluation of novel carbazole derivatives as potent DNMT1 inhibitors with reasonable PK properties

The DNA methyltransferases (DNMTs) were found in mammals to maintain DNA methylation. Among them, DNMT1 was the first identified, and it is an attractive target for tumour chemotherapy. DC_05 and DC_517 have been reported in our previous work, which is non-nucleoside DNMT1 inhibitor with low micromolar IC₅₀ values and significant selectivity towards other S-adenosyl-_L-methionine (SAM)-dependent protein methyltransferases. In this study, through a process of similarity-based analog searching, a series of DNMT1 inhibitors were designed, synthesized, and evaluated as anticancer agents. SAR studies were conducted based on enzymatic assays. And most of the compounds showed strong inhibitory activity on human DNMT1, especially WK-23 displayed a good inhibitory effect on human DNMT1 with an IC₅₀ value of 5.0 µM. Importantly, the pharmacokinetic (PK) profile of WK-23 was obtained with quite satisfying oral bioavailability and elimination half-life. Taken together, WK-23 is worth developing as DNMT1-selective therapy for the treatment of malignant tumour.     

谭清苏铁性别相关的RAPD标记研究

以谭清苏铁(Cycas tanqingii D.Y.Wang)雌雄植株半年生羽叶为材料,用优化的CTAB法分别提取其全基因组DNA,进行RAPD单因子梯度实验和正交实验以优化扩增条件。应用160个RAPD随机引物检测基因组DNA,雌雄植株均扩增出1450多条带,其中引物S0465扩增出与谭清苏铁雌株高度相关的RAPD标记,其大小约为500bp,该标记与雄株没有关联。     

Hemin with Peroxidase Activity Can Inhibit the Oxidative Damage Induced by Ultraviolet A

Excessive reactive oxygen species (ROS), a highly reactive substance that contains oxygen, induced by ultraviolet A (UVA) cause oxidative damage to skin. We confirmed that hemin can catalyze the reaction of tyrosine (Tyr) and hydrogen peroxide (H₂O₂). Catalysis was found to effectively reduce or eliminate oxidative damage to cells induced by H₂O₂ or UVA. The scavenging effects of hemin for other free-radical ROS were also evaluated through pyrogallol autoxidation, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·)-scavenging assays, and phenanthroline–Fe²⁺ assays. The results show that a mixture of hemin and tyrosine exhibits strong scavenging activities for H₂O₂, superoxide anion (O₂⁻·), DPPH·, and the hydroxyl radical (·OH). Furthermore, the inhibition of oxidative damage to human skin keratinocyte (HaCaT) cells induced by H₂O₂ or UVA was evaluated. The results show that catalysis can significantly reduce the ratio of cell apoptosis and death and inhibit the release of lactate dehydrogenase (LDH), as well as accumulation of malondialdehyde (MDA). Furthermore, the resistance to apoptosis was found to be enhanced. These results show that the mixture of hemin and tyrosine has a significantly protective effect against oxidative damage to HaCaT cells caused by UVA, suggesting it as a protective agent for combating UVA damage.     

The mode of action of thidiazuron: auxins,indoleamines, and ion channels in the regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.

The biochemical mechanisms underlying thidiazuron (TDZ)-induced regeneration in plant cells have not been clearly elucidated. Exposure of leaf explants of Echinacea purpurea to a medium containing TDZ results in undifferentiated cell proliferation and differentiated growth as mixed shoot organogenesis and somatic embryogenesis. The current studies were undertaken to determine the potential roles of auxin, indoleamines, and ion signaling in the dedifferentiation and redifferentiation of plant cells. E. purpurea leaf explants were found to contain auxin and the related indoleamine neurotransmitters, melatonin, and serotonin. The levels of these endogenous indoleamines were increased by exposure to TDZ associated with the induction of regeneration. The auxin-transport inhibitor 2,3,5-triiodobenzoic acid and auxin action inhibitor, p-chlorophenoxyisobutyric acid decreased the TDZ-induced regeneration but increased concentrations of endogenous serotonin and melatonin. As well, inhibitors of calcium and sodium transport significantly reduced TDZ-induced morphogenesis while increasing endogenous indoleamine content. These data indicate that TDZ-induced regeneration is the manifestation of a metabolic cascade that includes an initial signaling event, accumulation, and transport of endogenous plant signals such as auxin and melatonin, a system of secondary messengers, and a concurrent stress response.     

黄河三角洲垦利县夏季土壤水盐空间变异及土壤盐分微域特征

黄河三角洲作为我国重要的后备土地资源区,土壤盐渍化问题突出,切实掌握季节性土壤水盐状况及其微域特征是该区土壤盐渍化防控和土地资源高效利用的重要基础。选择黄河三角洲垦利县,通过野外调查实测与室内化验分析获取土壤水盐含量数据,利用统计分析、GIS空间插值、实地观测与数据分析对比等方法,分析了研究区夏季土壤水盐状况及其微域变异规律。结果显示:研究区夏季土壤水盐含量总体较高,含盐量以中度盐渍化为主,随着土层深度的增加含盐量呈上升趋势,且各层土壤含盐量呈显著正相关性;含盐量较高的地区主要分布在该区东北部和中东部,含盐量较低的地区主要分布在西南部和中部;土壤含盐量从大到小的植被类型依次为光板地→碱蓬→高粱→芦苇→茅草→水稻→棉花→玉米;土壤盐分微域变化特征明显,含盐量受距路边远近、不同耕作措施、地形部位、植被群落等因素影响较大,表现出微域规律性和复杂性。该研究基本摸清了研究区夏季时相的土壤水盐状况及其微域特征,为黄河三角洲农作物栽培管理及土壤资源可持续利用提供了科学依据。