α-酮戊二酸依赖型双加氧酶催化特性及反应耦联辅因子对其催化羟基化反应的影响

【目的】以重组大肠杆菌表达的枯草芽孢杆菌(Bacillus subtilis)L-异亮氨酸双加氧酶(L-isoleucine dioxygenase,IDO)为研究对象,考察其催化L-异亮氨酸(L-Ile)羟基化反应的影响因素,构建IDO催化合成羟基氨基酸的反应体系。【方法】通过Ni-NTA亲和层析法从重组大肠杆菌(Escherichia coli)BL21/p ET28a-ido中纯化获得重组IDO,以L-Ile为底物,考察重组IDO催化羟基化反应的影响因素,并进一步针对耦联反应优化α-酮戊二酸(α-KG)在重组IDO酶促转化体系中的添加浓度。【结果】基于重组IDO催化L-Ile羟基化的活性测定,计算该酶Km为0.247 mmol/L,kcat为1.260 s-1,kcat/Km为5.101 L/(mmol·s),与其他同源酶动力学参数比较分析表明,重组IDO的底物亲和性及催化效率较高。重组IDO催化反应的最适温度为20°C、最适p H为7.0;在35°C以下较为稳定;反应体系中Fe2+最适浓度为1 mmol/L。重组IDO可催化不同L-氨基酸反应,对L-异亮氨酸、L-正亮氨酸、L-甲硫氨酸的活性较高。通过优化α-KG浓度,反应体系中添加30 mmol/Lα-KG时,可将底物浓度提高至70 mmol/L,产物4-羟基异亮氨酸(4-HIL)的摩尔产率达66.20%,表明α-KG作为反应耦联辅因子,其浓度对重组IDO催化L-Ile羟基化具有显著影响。【结论】重组IDO的底物亲和性、催化效率、最适催化条件、稳定性等基本性质有利于催化L-Ile羟基化反应。在其催化反应体系中,α-KG作为反应耦联辅因子,对酶促转化效果影响显著。研究结果为4-HIL及其他羟基氨基酸的酶促转化提供了研究基础。     

盐生荒漠地表水热与二氧化碳通量的季节变化及驱动因素

以古尔班通古特沙漠南缘原始盐生荒漠为对象,利用涡度相关法,对原始盐生荒漠地表水热、二氧化碳通量进行了连续观测,对通量的季节变化、浅层土壤水分条件改变对盐生荒漠植物群落水汽、二氧化碳通量以及水分利用效率的影响进行了系统的分析.结果表明:净辐射通量、潜热通量和二氧化碳通量都具有明显的季节变化趋势,而显热通量的季节变化不明显.在有效能量的分配上,显热通量是有效能量的主要输出项.在降水影响期和非影响期,二氧化碳通量没有明显的变化;而在非降水影响期潜热通量明显降低,表明土壤水分处于亏缺状态,但二氧化碳通量并没有降低的趋势,而与前期保持高度的一致性.以此可以推断,该荒漠盐生植物群落并不以降水为主要水分来源,降水后水汽通量和二氧化碳通量变化的不一致性是该原始盐生荒漠独特植物特征决定的.降水影响期原始盐生荒漠植物群落的水分利用效率低于非影响期,是由于降水后土壤蒸发迅速增加,而植物蒸腾与光合并未随之增加造成的.     

Genetic validation of Aspergillus fumigatus phosphoglucomutase as a viable therapeutic target in invasive aspergillosis

Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a protective structure that is absent from human cells and is a potential target for antifungal treatments. Glucan is synthesized from the donor uridine diphosphate glucose, with the conversion of glucose-6-phosphate to glucose-1-phosphate by the enzyme phosphoglucomutase (PGM) representing a key step in its biosynthesis. Here, we explore the possibility of selectively targeting A. fumigatus PGM (AfPGM) as an antifungal treatment strategy. Using a promoter replacement strategy, we constructed a conditional pgm mutant and revealed that pgm is required for A. fumigatus growth and cell wall integrity. In addition, using a fragment screen, we identified the thiol-reactive compound isothiazolone fragment of PGM as targeting a cysteine residue not conserved in the human ortholog. Furthermore, through scaffold exploration, we synthesized a para-aryl derivative (ISFP10) and demonstrated that it inhibits AfPGM with an IC₅₀ of 2 μM and exhibits 50-fold selectivity over the human enzyme. Taken together, our data provide genetic validation of PGM as a therapeutic target and suggest new avenues for inhibiting AfPGM using covalent inhibitors that could serve as tools for chemical validation.     

Understanding pathogen–host interplay by expression profiles of lncRNA and mRNA in the liver of Echinococcus multilocularis-infected mice

Almost all Echinococcus multilocularis (Em) infections occur in the liver of the intermediate host, causing a lethal zoonotic helminthic disease, alveolar echinococcosis (AE). However, the long non-coding RNAs (lncRNAs) expression profiles of the host and the potential regulatory function of lncRNA during Em infection are poorly understood. In this study, the profiles of lncRNAs and mRNAs in the liver of mice at different time points after Em infection were explored by microarray. Thirty-one differentially expressed mRNAs (DEMs) and 68 differentially expressed lncRNAs (DELs) were found continuously dysregulated. These DEMs were notably enriched in “antigen processing and presentation”, “Th1 and Th2 cell differentiation” and “Th17 cell differentiation” pathways. The potential predicted function of DELs revealed that most DELs might influence Th17 cell differentiation and TGF-β/Smad pathway of host by trans-regulating SMAD3, STAT1, and early growth response (EGR) genes. At 30 days post-infection (dpi), up-regulated DEMs were enriched in Toll-like and RIG-I-like receptor signaling pathways, which were validated by qRT-PCR, Western blotting and downstream cytokines detection. Furthermore, flow cytometric analysis and serum levels of the corresponding cytokines confirmed the changes in cell-mediated immunity in host during Em infection that showed Th1 and Th17-type CD4⁺ T-cells were predominant at the early infection stage whereas Th2-type CD4⁺ T-cells were significantly higher at the middle/late stage. Collectively, our study revealed the potential regulatory functions of lncRNAs in modulating host Th cell subsets and provide novel clues in understanding the influence of Em infection on host innate and adaptive immune response.     

猪肌肉的转录组学研究进展

猪在中国的畜牧业及人类疾病的研究中有着不可或缺的作用,一方面猪提供了人类日常所需的肉类食物,另一方面在人类的器官移植方面的研究甚为广泛.因此,对于猪的各方面的研究都较为广泛,但在肌肉mRNA转录组方面涉及不深.转录组包括编码RNA和非编码RNA,根据测序结果,通过对不同品种的同位基因进行比较,用来分析相互之间的差异,进...     

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.     

浙江宁波镇海工业园区昆虫多样性研究

于2008年7-11月,对宁波镇海工业园区的昆虫群落多样性进行调查,选取林地、农田、人工苗圃、生态园和工厂区5种具有代表性的生境类型,每月对该地区的昆虫种类及数量进行抽样统计和数据分析.结果表明:该地共有昆虫284种,分属13目108科,其中优势类群为同翅目、直翅目、半翅目、鞘翅目和鳞翅目.在不同功能类群中,从个体数看...