Regulation of cyp26a1 on Th17 cells in mouse peri‐implantation

Cytochrome P450 26A1 (cyp26a1) is expressed in the mouse uterus during peri‐implantation. The repression of this protein is closely associated with a reduction in implantation sites, suggesting a specific role for cyp26a1 in pregnancy and prompting questions concerning how a metabolic enzyme can generate this distinct outcome. To explore the effective downstream targets of cyp26a1 and confirm if its role in peri‐implantation depends on its metabolic substrate RA (retinoic acid), we characterized the changes in the peripheral blood, spleen and uterine implantation sites using the cyp26a1 gene vaccine constructed before. Flow cytometry results showed a significant increase in CD4⁺RORγt⁺ Th17 cells in both the peripheral blood and spleen in the experimental group. The expression of RORγt and IL‐17 presented the Th17 cells reduction in uterus followed by the suppression of cyp26a1 expression. For greater certainty, cyp26a1 antibody blocking model and RNA interference model were constructed to determine the precise target immune cell group. High performance liquid chromatography results showed a significant increase in uterine at‐RA followed by the immunization of cyp26a1 gene vaccine. Both the ascertain by measuring RARα protein levels in peri‐implantation uterus after gene vaccine immunization and researches using the specific agonist and antagonist against RARα suggested that RARα may be the main RA receptor for signal transduction. These results provided more evidence for the signal messenger role of RA in cyp26a1 regulation from the other side. Here, we showed that the cyp26a1‐regulated Th17 cells are dependent on at‐RA signalling, which is delivered through RARα in mouse peri‐implantation.     

Murine folliculogenesis in vitro is stage-specifically regulated by insulin via the Akt signaling pathway

Folliculogenesis is a critical process during mammalian development, but little is known about the mechanisms underlying primordial follicular formation, growth, and activation. The objective of this study was to assess the effect of continuous insulin exposure on folliculogenesis in 16.5 dpc and 3 dpp mouse ovaries in vitro. The results demonstrated that 5 μg/ml insulin promotes the phosphorylation of Akt and the primordial follicular assembly and activation process via the InsR/PI3K/Akt signaling pathway in the 16.5 dpc fetal mouse ovaries cultured in vitro. However, the presence of 5 μg/ml insulin represses the phosphorylation of Akt and the follicular assembly and activation process in the 3 dpp mouse ovaries cultured in vitro. Furthermore, in vitro cultures of 3 dpp mouse ovaries with insulin can repress the expression of Pten. In conclusion, folliculogenesis was stage-specifically regulated by insulin via the Akt signaling pathway in vitro.     

Hydrogen Improves Glycemic Control in Type1 Diabetic Animal Model by Promoting Glucose Uptake into Skeletal Muscle

Hydrogen (H₂) acts as a therapeutic antioxidant. However, there are few reports on H₂ function in other capacities in diabetes mellitus (DM). Therefore, in this study, we investigated the role of H₂ in glucose transport by studying cultured mouse C2C12 cells and human hepatoma Hep-G2 cells in vitro, in addition to three types of diabetic mice [Streptozotocin (STZ)-induced type 1 diabetic mice, high-fat diet-induced type 2 diabetic mice, and genetically diabetic db/db mice] in vivo. The results show that H₂ promoted 2-[¹⁴C]-deoxy-d-glucose (2-DG) uptake into C2C12 cells via the translocation of glucose transporter Glut4 through activation of phosphatidylinositol-3-OH kinase (PI3K), protein kinase C (PKC), and AMP-activated protein kinase (AMPK), although it did not stimulate the translocation of Glut2 in Hep G2 cells. H₂ significantly increased skeletal muscle membrane Glut4 expression and markedly improved glycemic control in STZ-induced type 1 diabetic mice after chronic intraperitoneal (i.p.) and oral (p.o.) administration. However, long-term p.o. administration of H₂ had least effect on the obese and non-insulin-dependent type 2 diabetes mouse models. Our study demonstrates that H₂ exerts metabolic effects similar to those of insulin and may be a novel therapeutic alternative to insulin in type 1 diabetes mellitus that can be administered orally.     

弗氏志贺菌5a 型 M90T 株 ClpB 蛋白在大肠杆菌中的融合表达和纯化

目的：构建弗氏志贺菌cZp8基因的原核表达质粒,在大肠杆菌中表达后,纯化带T7标签的ClpB蛋白。方法与结果：PCR扩增得到线性化表达载体pET24a与2574bp的c加曰基因片段,利用不依赖连接反应的克隆法（LIC）进行克隆,得到重组质粒pET-ClpB,转入大肠杆菌BL21（DE3）中进行诱导表达,通过一系列条件优化,确定可溶性表达条件为在30℃下、用1mmol／LIPTG诱导2h;利用抗T7单克隆抗体琼脂糖珠进行亲和纯化,得到了纯度很高的相对分子质量为95×10^3的ClpB-T7融合蛋白。结论：实现了ClpB-T7在大肠杆菌中的可溶性表达,并纯化获得了高纯度的融合蛋白。     

串联亲和纯化原核表达载体的构建

【目的】构建串联亲和纯化原核表达载体,用于研究细菌中(生理状态或接近生理条件下的)蛋白-蛋白相互作用。【方法】设计并合成两条串联亲和标签序列,分别可以在靶蛋白N端和C端融合Protein G和链亲和素结合肽(Streptavidin binding peptide,SBP)标签;以pUC18载体为骨架,去除原有的阻遏蛋白基因,构建组成型表达载体pNTAP和pCTAP。【结果】成功构建N端和C端标签表达载体pNTAP和pCTAP,它们在大肠杆菌(Escherichia coli)BL21(DE3)、肠出血性大肠杆菌O157:H7和痢疾杆菌福氏5型M90T菌株中都可以实现表达。【结论】本实验构建的两个串联亲和纯化表达载体可以在部分革兰氏阴性细菌中表达,为研究细菌内蛋白-蛋白相互作用及致病菌毒力蛋白的作用机制奠定了基础。     

A co-culture system with preantral follicular granulosa cells in vitro induces meiotic maturation of immature oocytes

Development of technologies to mature oocytes in vitro is important for in vitro fertilization research. Here, we investigated the ability of preantral follicular granulosa cells (PAGCs) to restrain apoptosis and to promote the growth and meiotic resumption of immature murine oocytes in vitro. The oocytes of 55–65 μm derived from 12 to 14 days old juvenile mice were co-cultured with PAGCs in vitro. The results showed that the oocytes co-cultured with PAGCs for 7 days grew faster and 14.6% of immature oocytes were able to complete the first meiotic division and arrive at the MII stage. 71 oocytes co-cultured with PAGCs were fertilized and 16 embryos were able to form morula-blastocysts. Following the co-culture of immature oocytes with/without PAGCs for 7 days, the percentage of apoptotic oocytes were 33.5 and 51.4%, respectively (p < 0.01). Furthermore, the inhibition of apoptosis was communicated between oocytes and PAGCs through the GDF9-PI3 K-Akt signaling pathway. In conclusion, the co-culture with PAGCs has a beneficial effect on the growth and maturation of immature oocytes.     

Effect of Abandonment on Diversity and Abundance of Free-Living Nitrogen-Fixing Bacteria and Total Bacteria in the Cropland Soils of Hulun Buir,Inner Mongolia

In Inner Mongolia, steppe grasslands face desertification or degradation because of human over activity. One of the reasons for this condition is that croplands have been abandoned after inappropriate agricultural management. The soils in these croplands present heterogeneous environments in which conditions affecting microbial growth and diversity fluctuate widely in space and time. In this study, we assessed the molecular ecology of total and free-living nitrogen-fixing bacterial communities in soils from steppe grasslands and croplands that were abandoned for different periods (1, 5, and 25 years) and compared the degree of recovery. The abandoned croplands included in the study were natural restoration areas without human activity. Denaturing gradient gel electrophoresis and quantitative PCR (qPCR) were used to analyze the nifH and 16S rRNA genes to study free-living diazotrophs and the total bacterial community, respectively. The diversities of free-living nitrogen fixers and total bacteria were significantly different between each site (P<0.001). Neither the total bacteria nor nifH gene community structure of a cropland abandoned for 25 years was significantly different from those of steppe grasslands. In contrast, results of qPCR analysis of free-living nitrogen fixers and total bacteria showed significantly high abundance levels in steppe grassland (P<0.01 and P<0.03, respectively). In this study, the microbial communities and their gene abundances were assessed in croplands that had been abandoned for different periods. An understanding of how environmental factors and changes in microbial communities affect abandoned croplands could aid in appropriate soil management to optimize the structures of soil microorganisms.     

利用相似致病菌中保守的致病菌特有基因预测新的毒力相关基因

目的:在致病机制相似的致病菌中寻找保守的致病菌特有基因,预测新的毒力相关基因。方法:首先选取致病机制相似的致病菌EHEC与EPEC,利用本实验室构建的包含115 152条致病菌特有基因片段的数据库进行本地Blast,得到致病菌特有基因,对致病菌特有基因在相似致病菌中的保守性进行分析,得到新的可能的毒力相关基因。结果:在6株EHEC菌中找到95条保守的致病菌特有基因,其中大部分为已知的毒力相关基因,还有许多可能的毒力相关基因;在9株相似致病菌(EHEC、EPEC)中找到10条保守的致病菌特有的蛋白基因,其中9条为已知的致病相关基因,1条为可能的致病相关基因。结论:应用本方法可以发现新的毒力基因,为后续对致病菌致病机制的实验研究奠定了基础。     

Dechlorination of Chloral Hydrate Is Influenced by the Biofilm Adhesin Protein LapA in Pseudomonas putida LF54

LapA is the largest surface adhesion protein of Pseudomonas putida that initiates biofilm formation. Here, by using transposon insertion mutagenesis and a conditional lapA mutant, we demonstrate for the first time that LapA influences chloral hydrate (CH) dechlorination in P. putida LF54.     

Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase

Mitochondria are the major organelles that produce reactive oxygen species (ROS) and the main target of ROS-induced damage as observed in various pathological states including aging. Production of NADPH required for the regeneration of glutathione in the mitochondria is critical for scavenging mitochondrial ROS through glutathione reductase and peroxidase systems. We investigated the role of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) in controlling the mitochondrial redox balance and subsequent cellular defense against oxidative damage. We demonstrate in this report that IDPm is induced by ROS and that decreased expression of IDPm markedly elevates the ROS generation, DNA fragmentation, lipid peroxidation, and concurrent mitochondrial damage with a significant reduction in ATP level. Conversely, overproduction of IDPm protein efficiently protected the cells from ROS-induced damage. The protective role of IDPm against oxidative damage may be attributed to increased levels of a reducing equivalent, NADPH, needed for regeneration of glutathione in the mitochondria. Our results strongly indicate that IDPm is a major NADPH producer in the mitochondria and thus plays a key role in cellular defense against oxidative stress-induced damage.