Hypoxia‐induced miR‐3677‐3p promotes the proliferation,migration and invasion of hepatocellular carcinoma cells by suppressing SIRT5

Hepatocellular carcinoma (HCC), with life‐threatening malignant behaviours, often develops distant metastases and is the fourth most common primary cancer in the world, having taken millions of lives in Asian countries such as China. The novel miR‐3677‐3p is involved in a high‐expression‐related poor prognosis in HCC tissues and cell lines, indicating oncogenesis functions in vitro and in vivo. Initially, we confirmed the inhibition of proliferation, migration and invasion in miR‐3677‐3p knock‐down MHCC‐97H and SMMC‐7721 cell lines, which are well known for their high degree of invasiveness. Then, we reversed the functional experiments in the low‐miR‐3677‐3p‐expression Hep3B cell line via overexpressing miR‐3677‐3p. In nude mice xenograft and lung metastasis assays, we found suppressor behaviours, smaller nodules and low density of organ spread, after injection of cells transfected with shRNA‐miR‐3677‐3p. A combination of databases (Starbase, TargetScan and MiRgator) illustrated miR‐3677‐3p targets, and it was shown to suppress the expression of SIRT5 in a dual‐luciferase reporter system. To clarify the conclusions of previous ambiguous research, we up‐regulated SIRT5 in Hep3B cells, and rescue tests were established for confirmation that miR‐3677‐3p suppresses SIRT5 to enhance the migration and invasion of HCC. Interestingly, we discovered hypoxia‐induced miR‐3677‐3p up‐regulation benefited HCC malignancy and invasiveness. In conclusion, the overexpression of miR‐3677‐3p mediated SIRT5 inhibition, which could increase proliferation, migration and invasion of HCC in hypoxic microenvironments.     

猕猴桃RNA提取与RT-PCR

为了从富含多糖和多酚等物质的猕猴桃幼叶中提取和分离出高质量的RNA,用多个不同品种的猕猴桃叶为材料,比较了3种不同的RNA提取方法所提取的总RNA。结果表明,用胍-酚酸-DEPC法提取的RNA质量最好,提取率达到682.9~780.8μg?g(FW),其R值(A260?A280)接近1.90。用所提取的RNA样品进行RT-PCR,其扩增产物在琼脂糖凝胶上出现明显清晰的扩增cDNA带,说明RNA样品在纯度和浓度上都可以满足PCR等分子生物学实验的基本要求。     

Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure

This study investigated how Cd exposure affected oxidative biomarkers in Japanese flounder, Paralichthys olivaceus, at early life stages (ELS). Fish were exposed to waterborne Cd (0–48 µg L^− 1) from embryonic to juvenile stages for 80 days. Growth, Cd accumulation, activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione S-transferase (GST, EC 2.5.1.18), and levels of glutathione (GSH) and lipid peroxidation (LPO) were investigated at three developmental stages. Flounder growth decreased and Cd accumulation increased with increasing Cd concentration. In metamorphosing larvae, CAT and SOD activities were inhibited and GSH level was elevated, while LPO was enhanced by increasing Cd concentrations. CAT and GST activities of settling larvae were inhibited but GSH level was elevated at high Cd concentrations. In juveniles, SOD activity and LPO level were increased but GST activity was inhibited as Cd concentration increased. Antioxidants in flounder at ELS were able to develop ductile responses to defend against oxidative stress, but LPO fatally occurred due to Cd exposure. These biochemical parameters could be used as effective oxidative biomarkers for evaluating Cd contamination and toxicity in marine environments: CAT, SOD, GSH, and LPO for metamorphosing stage; CAT, GSH, and GST for settling stage; and SOD, GST, and LPO for juvenile stage.     

Enhancement of keratinolytic activity of a thermophilic subtilase by improving its autolysis resistance and thermostability under reducing conditions

WF146 protease, a thermophilic subtilase from thermophile Bacillus sp. WF146, suffers excessive autolysis in the presence of reducing agents. In this report, two autolytic sites of WF146 protease were modified by site-directed mutagenesis. The introduction of prolines into the autolytic sites increased the autolysis resistance of the enzyme under reducing conditions. The double mutant N63P/A66P displayed a 2.8-fold longer half-life at 80°C and higher hydrolytic activities than wild-type enzyme toward soluble (casein) and insoluble (keratin azure) substrates at high temperatures. In the presence of reducing agents, N63P/A66P was able to degrade feather at 80°C (∼3 h), with hydrolysis efficiency comparable to that of proteinase K at 50°C (∼24 h). Meanwhile, the mutant N63P/A66P had the ability to hydrolyze PrP^Sc-like prion protein at high temperatures. In virtue of these properties, N63P/A66P is of great interest to be used in recycling of keratinous wastes, such as feather, and disinfection of medical apparatus. In addition, our study may provide useful information needed to explore keratinolytic potential of thermophilic subtilases, even if they are produced by non-keratinolytic microorganisms.     

水稻XIOsPR10蛋白的原核表达及其RNase活性研究

PR10(病程相关蛋白10)类蛋白与植物的抵御外来病害及系统获得性抗性(SAR)有着紧密联系,而且许多PR10类蛋白都具有RNase活性,并通过这种活性抵抗外源病害。根据获得的XIOsPR10基因,将其构建到pET28a中,通过原核表达及磁珠纯化获得目的蛋白,通过RNA消化实验证实XIOsPR10重组蛋白具有RNase活性,进一步揭示了XIOsPR10蛋白的功能。     

Synergistic interactions of arbuscular mycorrhizal fungi and rhizobia promoted the growth of <Emphasis Type="Italic">Lathyrus sativus</Emphasis> under sulphate salt stress

In order to examine the influence of microsymbionts on plants, arbuscular mycorrhizal (AM) fungi and rhizobia were used to examine the growth of Lathyrus sativus under sulphate salt stress. Seedlings of L. sativus were inoculated with a combination of selected microsymbionts. Plants were grown under greenhouse conditions with five Na₂SO₄ concentrations (0, 1%, 2%, 3% and 4% (weight : weight)). The inoculations combinations used were the AM fungus, Glomus mosseae and/or the rhizobium, Mesorhizobium mediterraneum. The results showed that sulphate salinity inhibited plant growth and biomass production. However, compared with the control treatments, dual-inoculation of G. mosseae and M. mediterraneum reduced the harmful influence of sulphate salinity. Parmeters including plant height, the extent of AM colonization, total biomass, nodules biomass, P concentration, N concentration and proline concentration confirmed that dual inoculation plays a vital role in promoting the growth of L. sativus under sulphate salt stress. The results suggested that the use of this dual inoculation could be exploited in grassland plantation establishment and in pastoral ecosystem reclamation programmes in arid and semi-arid areas subject to moderate salt contamination.     

天然林生态系统稳定性与病虫害干扰——调控与被调控

如何评价天然林中原生的昆虫、病原的生态功能是制定病虫害防治措施首先要明确的问题之一。依据天然林生态系统稳定性与病虫害干扰之间关系的最新研究进展,系统阐述了当前有关天然林调控病虫害爆发的多样性-稳定性假说和联合抗性假说,指出联合抗性假说的不确定性以及多样性-稳定性假说的局限性。昆虫、病原在天然林演替过程对系统整体结构、健康、稳定以及可持续性上不但发挥着重要的生态调控功能作用,在有些情况下甚至加速或改变系统的演替途径。这种生物干扰目前在林业发达国家的天然林管理策略中被视为有益的干扰元素,由此提出将模拟自然干扰作为今后天然林管理策略一项指导方针。同时指出我国对此的认识不仅较国外林业发达国家晚,而且在实践中也未能有效地整合在森林生态系统健康管理的实践中,因此这种对天然林生物干扰的新认识有助于未来我国天然林病虫害生态控制治理决策的制定。     

P277多肽融合热休克蛋白65提高抗1型糖尿病的作用

为了提高P277肽抗1型糖尿病的作用, 把P277肽融合在卡介苗热休克蛋白65的C端, 构建了pET28a- HSP65-P277高效表达载体, 在大肠杆菌中高效可溶性表达。利用硫酸铵分级沉淀、阴离子交换柱层析分离纯化了融合蛋白HSP65-P277。使用HSP65-P277在没有任何佐剂存在的情况下免疫非肥胖性糖尿病(NOD)小鼠, 通过三次腹腔注射, 每月收集被免疫动物的血清, 血糖浓度用自动生化分析仪测定。结果显示HSP65-P277免疫组小鼠血糖平均值及糖尿病的累积发病率和其余组相比均有显著差异(P<0.01), 融合蛋白HSP65-P277抗NOD小鼠糖尿病的作用显著高于单独的P277和HSP65。为进一步开发能用于临床的1型糖尿病疫苗提供了良好的设计思路, HSP65-P277极有可能进一步发展成为新的抗I型糖尿病的疫苗。     

Induction,purification, and characterization of a thermo and pH stable laccase from Abortiporus biennis J2 and its application on the clarification of litchi juice

A fungus J2 producing laccase with high yield was screened in soils and identified as Abortiporus biennis. The production of laccase was induced by 0.1 mM Cu²⁺, 0.1 mM tannic acid, and 0.5 M ethanol. The laccase from Abortiporus biennis J2 was purified to electrophoretic homogeneity by a couple of steps. The N-terminal amino acid sequence of the enzyme was AIGPTADLNISNADI. The properties of the purified laccase were investigated. The result showed the laccase from Abortiporus biennis J2 is a thermo and pH stable enzyme. The laccase activity was inhibited by Hg²⁺, Cd²⁺, Fe²⁺, Ag⁺, Cu²⁺, and Zn²⁺, while promoted by Mg²⁺, Mn²⁺ at 10 mM level. Purified laccase was used to the clarification of litchi juice. After treatment with this laccase, the phenolic content of litchi juice had been found to be greatly reduced along with an increase in the clarity of the juice. The result indicated the potential of this laccase for application in juice procession.