Proteomics analysis reveals diabetic kidney as a ketogenic organ in type 2 diabetes

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. To date, the molecular mechanisms of DN remain largely unclear. The present study aimed to identify and characterize novel proteins involved in the development of DN by a proteomic approach. Proteomic analysis revealed that 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase 2 (HMGCS2), the key enzyme in ketogenesis, was increased fourfold in the kidneys of type 2 diabetic db/db mice. Consistently, the activity of HMGCS2 in kidneys and 24-h urinary excretion of the ketone body β-hydroxybutyrate (β-HB) were significantly increased in db/db mice. Immunohistochemistry, immunofluorescence, and real-time PCR studies further demonstrated that HMGCS2 was highly expressed in renal glomeruli of db/db mice, with weak expression in the kidneys of control mice. Because filtered ketone bodies are mainly reabsorbed in the proximal tubules, we used RPTC cells, a rat proximal tubule cell line, to examine the effect of the increased level of ketone bodies. Treating cultured RPTC cells with 1 mM β-HB significantly induced transforming growth factor-β1 expression, with a marked increase in collagen I expression. β-HB treatment also resulted in a marked increase in vimentin protein expression and a significant reduction in E-cadherin protein levels, suggesting an enhanced epithelial-to-mesenchymal transition in RPTCs. Collectively, these findings demonstrate that diabetic kidneys exhibit excess ketogenic activity resulting from increased HMGCS2 expression. Enhanced ketone body production in the diabetic kidney may represent a novel mechanism involved in the pathogenesis of DN.     

Low Molecular Weight Heparin Ablates Lung Cancer Cisplatin-Resistance by Inducing Proteasome-Mediated ABCG2 Protein Degradation

Cancer side population (SP) cells, which are often referred to as cancer stem cells, are thought to be responsible for lung cancer chemotherapy resistance, and currently no drug can specifically target these cells. We hypothesize low-molecular-weight heparin (LMWH) may affect the biological properties of SP cells and could be used to clinically target these cells. To test this, SP cells were isolated from cisplatin (DDP)-resistant lung adenocarcinoma A549/DDP cells by flow cytometric sorting. Compared to non-SP cells, SP cells formed increased numbers of colonies in vitro, and had a 1000-fold increase in tumorigenicity in vivo. Proliferation and apoptosis assays demonstrated LMWH had no significant effect on lung SP cell proliferation or apoptosis. However, LMWH reduced lung SP cell colony formation ability and protein expression of the multidrug transporter, ABCG2, by FACS and western blot analyses without affecting its mRNA levels by RT-PCR. Consistently, immunohistochemistry stainings of ABCG2 in LMWH-treated tumor tissues were significantly reduced compared with those in controls. Further, we found proteasomal inhibitor MG132, but not lysosomal inhibitors leupeptin and pepstatin A, could restore ABCG2 protein levels in LMWH-treated SP cells. These suggest LMWH ablates lung SP cell chemoresistance by proteasome-mediated reduction of ABCG2 protein levels without affecting its mRNA levels. We also determined LMWH combined with cisplatin could overcome cisplatin-resistance and induced lung SP cells apoptosis both in vitro and in vivo. This study provides an experimental basis for using a combination of LMWH, which targets lung SP cells, with chemotherapy to improve lung cancer survival.     

Decoy engineering of the receptor-like cytoplasmic kinase StPBS1 to defend against virus infection in potato

Potato virus Y (PVY) is an important pathogen of potato (Solanum tuberosum). Although the PBS1–RPS5 immune system is well documented in Arabidopsis thaliana, it has not been reported in potato. In Arabidopsis, the bacterial effector AvrPphB cleaves AtPBS1 to trigger an immune response. Here, we show that the AvrPphB-triggered immune response is mediated by StPBS1, a close homologue of AtPBS1 in potato. However, downstream signalling of StPBS1 was mediated by unknown resistance (R) proteins other than potato orthologues of AtRPS5 and HvPBR1, which is important for HvPBS1 signalling in barley. Immune signalling of StPBS1 is mediated by the AvrPphB C-terminal cleavage domain and an STKPQ motif, in contrast to AtPBS1-mediated immunity in which both AvrPphB cleavage fragments and an SEMPH motif are essential. The cleavage sequence of AvrPphB in StPBS1 was replaced with that of the PVY NIa-Pro protease to obtain StPBS1^NIa. StPBS1^NIa overexpression potato displayed stronger immunity to PVY infection than did the StPBS1 transgenic lines. StPBS1^NIa was cleaved at the expected target site by NIa-Pro protease from PVY. Thus, we characterized the function of StPBS1 in potato immunity and provide a biotechnology control method for PVY via transformation of decoy-engineered StPBS1^NIa.     

高寒草甸植物物种丧失对草原毛虫的影响

植物多样性是调控食物网结构和生态系统功能最重要的生物因素, 植物多样性丧失深刻影响食草动物, 但由于小型食草动物种群数量波动明显、统计随机性较大等困难, 我们对植物多样性丧失如何影响小型食草动物依然知之甚少。基于在青藏高原高寒草甸设置的长期植物物种剔除试验, 本研究于2016-2020年7-8月连续调查了植物物种剔除各处理中草原毛虫(Gynaephora alpherakiif)的数量, 分析了植物物种及功能群丧失对草原毛虫的影响。结果表明, 虽然时空差异及统计随机性是影响草原毛虫数量变化的主要因素, 但植物物种剔除介导的群落差异对草原毛虫数量的影响依然显著: (1)在各观测时段, 优势种线叶嵩草(Kobresia capillifolia)的丧失导致群落中草原毛虫数量显著减少; 禾草类物种丧失也会减少草原毛虫数量, 但其影响仅在8月显著; (2)杂类草物种丧失通过增加群落中禾草物种多度, 可增加草原毛虫数量; 豆科物种丧失使莎草增多, 也会增加草原毛虫数量; (3)各植物功能群部分物种剔除并未显著影响草原毛虫数量。本研究证实了高寒草甸中草原毛虫数量会因优势植物嵩草和禾草的多度减少或禾草物种丧失而显著减少, 但群落总生物量、个体数和物种丰富度、豆科多度以及各功能群植物同比减少, 都对草原毛虫数量没有明显影响。这些结果说明在随机作用主导下, 植物群落中的特定功能群相对多度(而非物种多样性)变化深刻影响草原毛虫适合度, 进而影响生态系功能及服务; 未来生物多样性研究及草地虫害生物防控中应更多考虑统计随机性及植物功能多样性对小型食草动物的影响。     

基于近红外漫反射光谱快速测定淫羊藿蛋白质含量

采用近红外漫反射光谱技术对淫羊藿(Epimedium)的蛋白质含量进行快速且无损检测。近红外漫反射光谱经二阶导数处理、标准多元离散校正及主成分分析聚类处理后, 采用改进最小二乘法回归得到的定标模型预测效果最佳, 定标决定系数、交互验证标准差及交互验证相关系数分别为0.923、0.554和0.717。近红外光谱分析法的测定结果与用凯氏定氮法所得结果无显著差异, 两种方法测定值的相关性较高(R²=0.933 9)。重复性实验表明, 近红外光谱分析法的相对标准偏差为0.937%。该研究首次采用近红外光谱分析法测定了8种淫羊藿的蛋白质含量。该方法简便、精确, 在淫羊藿资源开发利用和药材质量控制方面具有参考意义。     

凹甲陆龟沙门氏菌的分离鉴定及毒力因子基因的PCR检测

从成都动物园因腹泻死亡的凹甲陆龟体内分离到一株病源菌,经选择性分离培养、生化试验、血清型鉴定等,确定该病源菌为D群肠炎沙门氏菌,其抗原结构式为1,9,12∶ g,m∶ -.该菌株对小鼠有较强的致病性,能引起人工感染小鼠大量死亡,且从其体内分离到相同特性的菌株;药物敏感性试验证实该分离菌对头孢曲松、氧氟沙星、卡拉霉素、多粘菌素、复方新诺明、呋喃唑酮等敏感,对链霉素、四环素、氯霉素等耐药;同时根据GenBank中已报道的沙门氏菌毒力因子ivnA和ivnE的基因序列,设计两对特异性引物对该菌进行PCR检测,结果在该菌中成功扩增并检测出ivnA和ivnE基因.本试验尝试了沙门氏菌的PCR检测方法,为该分子诊断技术的全面推广奠定了良好基础,也为今后该疾病的预防、诊断和治疗提供了科学的实验数据.     

水稻病程相关PR1家族蛋白质在叶片生长及与白叶枯病菌互作反应中的表达

病程相关(PR)蛋白质经常被用作抗病反应的分子标记。利用免疫印迹(WB)技术检测了7个PR1家族蛋白质在水稻(Oryza sativa)叶片生长及与白叶枯病菌互作反应过程中的表达,发现6个PR1家族蛋白质在叶片生长中有表达。检测PR1蛋白质在Xa21介导的抗白叶枯病过程中的表达,结果显示PR1#052、PR1#072、PR1#073和PR1#121四个蛋白质在抗病反应后期呈上调或诱导表达,PR1#071则表达下调。进一步比较它们在抗病、感病和对照(Mock)反应中的表达丰度,发现在抗病和感病反应中的变化幅度均明显大于对照反应,推测这些PR蛋白质在水稻-白叶枯病菌互作反应中发挥作用。另外,对PR1基因上游启动子区的cis元件进行了分析。该研究初步揭示了水稻PR1家族蛋白质的表达谱,为进一步了解PR1蛋白质的功能提供了线索。     

ROS-Ca is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis

The surfactin can inhibit proliferation and induce apoptosis in cancer cells. Moreover, surfactin can induce cell death in human breast cancer MCF-7 cells through mitochondrial pathway. However, the molecular mechanism involved in this pathway remains to be elucidated. Here, the reactive oxygen species (ROS) and Ca²⁺ on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis which induced by surfactin were investigated. It is found that surfactin evoked mitochondrial ROS generation, and the surfactin-induced cell death was prevented by N-acetylcysteine (NAC, an inhibitor of ROS). An increasing cytoplasmic Ca²⁺ concentration was detected in surfactin-induced MCF-7 apoptosis, which was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium). In addition, the relationship between ROS generation and the increase of cytoplasm Ca²⁺ was determined. The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential (ΔΨ_m). Then the cytoplasmic Ca²⁺ concentration increased in virtue of the changes of mitochondrial permeability, which was prevented by BAPTA-AM. Besides, cytochrome c (cyt c) was released from mitochondria to cytoplasm through the MPTP and activated caspase-9, eventually induced apoptosis. In summary, surfactin has notable anti-tumor effect on MCF-7 cells, however, there was no obvious cytotoxicity on normal cells.     

高脂血症兔球结膜微循环的改变

本实验用胆固醇粉喂家兔,造成实验性高脂血症,活体观察球结膜微循环的形态、流态及血管周围状态等１５项指标,并用球结膜微循环综合定量评价方法计算了综合积分值。处死后取球结膜进行组织学检查。结果表明：高脂血症家兔球结膜微循环有明显改变,主要为微血管形态异常、血流减慢、红细胞聚集、白微栓、静脉壁上有白色斑块等改变。球结膜微循环综合积分值明显增加,高于实验前。球结膜组织学检查发现上皮层下有泡沫细胞、血管壁增厚、有空泡、静脉内有血栓。虹膜睫状体内除有多数泡沫细胞外,还有胆固醇的菱形结晶。上述改变表明,高脂血症兔球结膜微循环有明显改变。血液有高凝、高聚倾向。黑茶中的茶色素有抗凝、抑制血小板聚集、促进纤溶等作用,口服黑茶可改善高脂血症兔球结膜微循环障碍。     

Conditioned medium from hypoxia-treated adipocytes renders muscle cells insulin resistant

Adipose tissue hypoxia is an early phenotype in obesity, associated with macrophage infiltration and local inflammation. Here we test the hypothesis that adipocytes in culture respond to a hypoxic environment with the release of pro-inflammatory factors that stimulate macrophage migration and cause muscle insulin resistance. 3T3-L1 adipocytes cultured in a 1% O2 atmosphere responded with a classic hypoxia response by elevating protein expression of HIF-1α. This was associated with elevated mRNA expression and peptide release of cytokines TNFα, IL-6 and the chemokine monocyte chemoattractant protein-1 (MCP-1). The mRNA and protein expression of the anti-inflammatory adipokine adiponectin was reduced. Conditioned medium from hypoxia-treated adipocytes (CM-H), inhibited insulin-stimulated and raised basal cell surface levels of GLUT4myc stably expressed in C2C12 myotubes. Insulin stimulation of Akt and AS160 phosphorylation, key regulators of GLUT4myc exocytosis, was markedly impaired. CM-H also caused activation of JNK and S6K, and elevated serine phosphorylation of IRS1 in the C2C12 myotubes. These effects were implicated in reducing propagation of insulin signaling to Akt and AS160. Heat inactivation of CM-H reversed its dual effects on GLUT4myc traffic in muscle cells. Interestingly, antibody-mediated neutralization of IL-6 in CM-H lowered its effect on both the basal and insulin-stimulated cell surface GLUT4myc compared to unmodified CM-H. IL-6 may have regulated GLUT4myc traffic through its action on AMPK. Additionally, antibody-mediated neutralization of MCP-1 partly reversed the inhibition of insulin-stimulated GLUT4myc exocytosis caused by unmodified CM-H. In Transwell co-culture, hypoxia-challenged adipocytes attracted RAW 264.7 macrophages, consistent with elevated release of MCP-1 from adipocytes during hypoxia. Neutralization of MCP-1 in adipocyte CM-H prevented macrophage migration towards it and partly reversed the effect of CM-H on insulin response in muscle cells. We conclude that adipose tissue hypoxia may be an important trigger of its inflammatory response observed in obesity, and the elevated chemokine MCP-1 may contribute to increased macrophage migration towards adipose tissue and subsequent decreased insulin responsiveness of glucose uptake in muscle.