F-box domain is involved in regulating the proliferation of MCF-7 cells by FBXO39 protein

AIM: To investigate the effect of F-box domain on the regulation of MCF-7 cell proliferation by FBXO39 protein. METHODS: The effect of F-box domain on the localization of FBXO39 protein in the MCF-7 cells was investigated. MCF-7 cell cDNA library was used as the template resource. The full-length cDNA sequence of FBXO39 was amplified by PCR method and subcloned into eukaryotic expression vector pEGFP-C2. The pEGFP-FBXO39ΔF (F-box domain deletion mutation) plasmid was successfully constructed with the template resource of pEGFP-FBXO39 plasmid. The recombinant plasmids were transfected into the MCF-7 cells, and then the expression of FBXO39 and FBXO39ΔF were determined by Western blot. The cellular localization of FBXO39 and FBXO39ΔF were observed by confocal microscopy. The localization of endogenous FBXO39 in the MCF-7 cells was detected by immunofluorescence staining. In addition, MTT and EdU assays were used to measure the cell proliferation, flow cytometry was used to measure the cell cycle distribution, and immunohistochemical staining was used to observe the expression of FBXO39 in the breast cancer and para-carcinoma tissues. RESULTS: The eukaryotic expression vector pEGFP-FBXO39 and pEGFP-FBXO39ΔF were constructed successfully. F-box domain had no effect on the cell localization of FBXO39. FBXO39 promoted MCF-7 cell proliferation but FBXO39ΔF did not. FBXO39 was highly expressed in the breast cancer tissues. CONCLUSION: F-box domain had no effect on the cellular localization of FBXO39 protein. However, it plays an important role in the biological function of FBXO39. FBXO39 may be related to breast cancer tumorigenesis.     

SPI凝胶颗粒制备及其Pickering高内相乳液特性研究

制备了大豆分离蛋白（Soy protein isolate，SPI）凝胶颗粒，并使用该颗粒制备了稳定的Pickering高内相乳液。通过粒径和ζ 电位测定、冷冻扫描电镜和光学显微镜观察以及外观分析，以流变特性为指标，对SPI凝胶颗粒和Pickering高内相乳液特性进行研究。结果表明，SPI凝胶颗粒的粒径和ζ 电位随pH值的变化而变化，当SPI凝胶颗粒pH值为4.0～5.0时，临近SPI等电点，ζ 电位的绝对值最小，此时凝胶颗粒相互聚集，不能成功制备稳定的高内相乳液。在pH值为 9.0时，大豆分离蛋白凝胶颗粒紧密结合在一起，呈凝胶网络状结构。在碱性条件下，蛋白质质量分数为1.00%、内相体积分数为78%～82%时，可以制备稳定的Pickering高内相乳液。通过增加内相体积分数，使大豆分离蛋白凝胶颗粒稳定的Pickering乳液体系分布更加均匀，不易发生聚集，形成更加致密稳定的多孔结构，且具有更强的弹性凝胶乳液特性。     

大鼠成熟的肺表面活性蛋白B的原核表达及纯化

为表达和纯化SP-B蛋白,先对SP-B基因进行稀有密码子优化,PCR反应获得SP-B片段,构建重组质粒pGEX4T-1/SP-B,转化到E.coli BL21(DE3)中诱导表达;用SDS-PAGE与Western blot进行检测;用GSTPrep FF 16/10对融合蛋白进行纯化。经双酶切鉴定证实质粒中插入基因长250bp,测序结果与大鼠SP-B cDNA序列相符;质粒转化后用IPTG进行诱导,在分子质量约34ku处出现1个新条带,与pGEX4T-1/SP-B蛋白预期大小一致,且该融合蛋白能可溶性表达并被纯化。结果表明成功构建了pGEX4T-1/SP-B重组质粒,表达、纯化得到GST/SP-B蛋白,为研究肺表面活性物质替代药物奠定了基础。     

高压电场干燥和热风干燥对马铃薯蛋白质二级结构的影响

以自然风干为对照组,采用SDS-PAGE凝胶电泳和圆二色光谱法研究了高压电场干燥(HVEF)和热风干燥对马铃薯蛋白质分子量的影响以及两种干燥方法对马铃薯蛋白质二级结构的影响。SDS-PAGE凝胶电泳结果显示:两种干燥方法对马铃薯蛋白质分子量基本不产生影响。圆二色光谱检测结果表明:经高压电场干燥后,马铃薯蛋白质的α-螺旋所占比例下降1.30%,β-折叠所占比例上升1.60%,β-转角所占比例上升0.30%,无规卷曲所占比例上升1.20%;经热风干燥后,马铃薯蛋白质的α-螺旋所占比例上升了12.50%,β-折叠所占比例下降了10.70%,β-转角所占比例上升了4.30%,无规卷曲所占比例下降4.40%。通过比较:HVEF对马铃薯蛋白质二级结构中α-螺旋、β-折叠、无规卷曲的作用趋势与热风干燥正好相反;如,热风干燥使α-螺旋的比例显著上升,而HVEF则使α-螺旋的比例下降,并且HVEF对马铃薯蛋白质二级结构的影响比热风干燥对马铃薯蛋白质二级结构的影响较小。     

拟南芥乙烯应答基因HLS1的原核表达及多克隆抗体制备

【目的】制备乙烯应答基因HLS1多克隆抗体,在过表达植物中检测HLS1的积累,为深入研究HLS1响应乙烯信号通路的分子机制奠定基础。【方法】构建pET28a-HLS1c原核表达载体,转化到大肠杆菌BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导获得重组蛋白HLS1c-His;镍柱亲和纯化重组蛋白后免疫家兔获得抗HLS1血清,利用抗原亲和纯化抗血清获得高纯度的HLS1多克隆抗体;用HLS1抗体和GFP抗体,检测原生质体瞬时转化体系中GFP-HLS1c融合蛋白的表达;用农杆菌蘸花法获得过表达GFP-HLS1g的转基因植物,用HLS1抗体和GFP抗体检测GFP-HLS1g融合蛋白的表达。【结果】成功构建了原核表达载体pET28a-HLS1c,并在大肠杆菌系统中诱导获得重组蛋白HLS1c-His,且多以包涵体形式存在。纯化的HLS1c-His多克隆抗体,能清晰检测到约80 ng原核表达的HLS1抗原。纯化的HLS1抗体不仅能检测到原生质体中瞬时表达的GFP-HLS1c融合蛋白,也能检测到过表达转基因植物株系中的GFP-HLS1g融合蛋白,并且与标签蛋白GFP对应抗体所检测到的特异性条带大小一致。【结论】成功制备了拟南芥HLS1蛋白多克隆抗体,为HLS1蛋白在植物发育中的功能研究奠定了基础。     

Correlation between Mnk2 protein expression and prognosis of patients with esophageal squamous cell carcinoma

AIM: To investigate the protein expression of mitogen-activated protein kinase-interacting kinase-2 (Mnk2) and its prognostic effect in the patients with resected esophageal squamous cell carcinoma (ESCC). METHODS: A total of 86 informative patients with surgically resected ESCC and 54 normal esophageal tissues were enrolled. Western blot and immunohistochemistry (IHC) were utilized to assess the protein expression of Mnk2, and its correlation with prognosis was statistically analyzed by the methods of Kaplan-Meier curve and Cox proportional hazard mode. RESULTS: The protein expression of Mnk2 was elevated in most of tumor tissues compared with the adjacent tissues. Clinicopathologic analysis showed that Mnk2 expression was significantly correlated with the TNM stage (P<0.05). Both disease-free survival (DFS) and overall survival (OS) of Mnk2 over-expression patients were shorter than those in Mnk2 negative expression group. Multivariate analysis confirmed that Mnk2 expression, as an independent and significant factor for both DFS and OS, predicted a poor prognosis of the patients with resected ESCC (P<0.05). CONCLUSION: The expression of Mnk2 was significantly related to the TNM stages, and might be a novel predictor for prognosis in ESCC.     

Can additional N fertiliser ameliorate the elevated CO2‐induced depression in grain and tissue N concentrations of wheat on a high soil N background?

Elevated CO₂ stimulates crop yields but leads to lower tissue and grain nitrogen concentrations [N], raising concerns about grain quality in cereals. To test whether N fertiliser application above optimum growth requirements can alleviate the decline in tissue [N], wheat was grown in a Free Air CO₂ Enrichment facility in a low‐rainfall cropping system on high soil N. Crops were grown with and without addition of 50–60 kg N/ha in 12 growing environments created by supplemental irrigation and two sowing dates over 3 years. Elevated CO₂ increased yield and biomass (on average by 25%) and decreased biomass [N] (3%–9%) and grain [N] (5%). Nitrogen uptake was greater (20%) in crops grown under elevated CO₂. Additional N supply had no effect on yield and biomass, confirming high soil N. Small increases in [N] with N addition were insufficient to offset declines in grain [N] under elevated CO₂. Instead, N application increased the [N] in straw and decreased N harvest index. The results suggest that conventional addition of N does not mitigate grain [N] depression under elevated CO₂, and lend support to hypotheses that link decreases in crop [N] with biochemical limitations rather than N supply.     

鸡白细胞介素2的克隆及其序列分析

旨在从分子生物学角度进一步研究鸡免疫刺激因子白细胞介素2(IL-2),探究IL-2蛋白结构。根据GenBank发表的IL-2序列设计引物扩增IL-2基因,将其克隆到p GM-T载体后进行鉴定,鉴定正确的pT-IL-2重组质粒,然后对其进行序列分析。应用生物信息学软件对IL-2序列进行跨膜区、磷酸化位点、氨基酸序列、开放阅读框进行分析和IL-2蛋白的三级结构进行预测。核苷酸序列测定结果表明:IL-2基因片段为430 bp,编码143个氨基酸。生物信息学分析结果表明:本实验获得的IL-2基因编码的蛋白质中含有1个丝氨酸和3个苏氨酸,这可能成为蛋白激酶磷酸化的位点。IL-2蛋白可能在5~28位含有跨膜区。其蛋白二级、三级结构预测结果显示其属于亲水性蛋白,多为α-螺旋、β-转角和N端无规卷曲结构。该结果为IL-2的分子生物学探究奠定了基础。     

苦瓜对白粉病的抗性与相关生理生化指标的关系

为探讨苦瓜抗白粉病的生理生化机制,以对白粉病不同抗性的4个苦瓜品系为材料,研究苗期感染白粉病菌后,苦瓜叶片生理生化指标的变化。结果表明:接种后,叶绿素和可溶性蛋白质量分数均呈先上升后降低的趋势;可溶性糖质量分数呈下降-上升-下降-上升的趋势;抗病品系的可溶性糖、叶绿素质量分数和过氧化物酶(POD)、多酚氧化酶(PPO)活性均高于或显著高于感病品系和高感品系;抗病品系的抗坏血酸(AsA)质量分数的上升和下降的幅度均小于感病品系和高感品系;接种后10~20d,抗坏血酸过氧化物酶(APX)活性表现为抗病品系感病品系高感品系。叶绿素、可溶性糖质量分数和POD、PPO活性与病情指数呈显著或极显著负相关。综上说明,白粉病菌侵染苦瓜后,抗病品系可通过保持较高的叶绿素质量分数,增加可溶性糖、可溶性蛋白、AsA质量分数及增强POD、PPO、APX活性来提高抗病性。叶绿素、可溶性糖质量分数和POD、PPO活性均可作为苦瓜对白粉病抗性早期鉴定的指标。     

Liraglutide ameliorates liver injury of type 2 diabetic mice via micro-RNA-33-AMPK pathway

AIM: To observe the effects of liraglutide on the level of microRNA-33 (miR-33) and the expression of AMP-activated protein kinase (AMPK) and apoptosis-related proteins in mice with type 2 diabetes mellitus (T2DM), and to explore its possible mechanism. METHODS: High-fat diet and intraperitoneal injection of streptozocin were used to establish the type 2 diabetic model in C57BL/6 mice. The mice were randomly divided into 4 groups (n=15):in control group, the normal mice were subcutaneously injected with equivalent volume of saline; in model group, the T2DM mice were subcutaneously injected with equivalent volume of saline; in low-and high-dose liraglutide treatment groups, the T2DM mice were subcutaneously injected with 100 and 200 μg·kg^-1·d^-1, respectively. After 4 weeks of administration, the levels of FBG, TG, TC, HDL-C, LDL-C, ALT and AST were determined. HE staining was used to observe the pathological changes of the liver tissues. The protein level of cleaved caspase-3 in the liver tissue was detected by the technique of immunofluorescence. The protein levels of p-AMPK/AMPK and apoptosis-related proteins were detected by Western blot. The expression of miR-33 in the liver tissues was detected by real-time PCR. RESULTS: Compared with model group, the contents of FBG, TG, TC, LDL-C, ALT and AST were decreased significantly, while the content of HDL-C was increased significantly in low-dose liraglutide group and high-dose liraglutide group (P<0.05). The protein levels of phosphorylated AMPK and Bcl-2 were up-regulated significantly, and the expression of cleaved caspase-3 was down-regulated significantly (P<0.05). The level of miR-33 was decreased significantly (P<0.01). CONCLUSION: Liraglutide alleviates liver injury in type 2 diabetic mice, and the mechanism may be associated with reducing the level of miR-33 and increasing the phosphorylation of AMPK in the liver tissues, thereby inhibiting hepatocyte apoptosis.