一个水稻NADPH氧化还原酶相似基因的克隆及表达分析

采用荧光差显(fluorescent differential display,FDD)技术,比较分析了干旱处理与未处理水稻叶片及根中的mRNA表达,并利用H. A. Yellow PAGE(含0.1% H. A. Yellow)再分离与大矩阵(macroarray)筛选相结合的方法,发现1个与拟南芥NADPH氧化还原酶高度相似(96%)的差异片段。该基因的cDNA全长为1423 bp,编码一个具有345个氨基酸残基的多肽。在正常情况下该基因表达量很低,而在干旱处理中高表达。讨论了干旱胁迫下NADPH氧化还原酶基因的可能功能。     

Roles of peroxidases and NADPH oxidases in the oxidative response of wheat (Triticum aestivum) to brown rust (Puccinia triticina) infection

The goal of this work was to establish which enzymes – peroxidases or NADPH oxidases – play the most important role in the resistance‐related oxidative burst response of wheat to infection by brown rust (Puccinia triticina). The expression of four peroxidases and two NADPH oxidases was analysed in the susceptible wheat cv. Thatcher and isogenic lines with different Lr resistance genes after pathogen inoculation. Of the peroxidases, TaPrx118 and TaPrx112 were induced several times more strongly than TaPrx103 and TaPrx107. The induction of peroxidases was more pronounced than that of NADPH oxidases. The patterns of peroxidase expression clearly differentiated moderately resistant from highly resistant lines and corresponded to oxidative response profiles. The possible involvement of peroxidases or NADPH oxidases was verified with enzyme‐specific inhibitors. The oxidative burst in the susceptible cv. Thatcher and in the lines TcLr24, TcLr25, TcLr9 was peroxidase‐dependent, while the response in line TcLr26 was NADPH‐oxidase‐dependent. It is postulated that class III peroxidases play a leading role in the formation of reactive oxygen species molecules during the response of wheat to pathogen infection. The results suggest a high level of redundancy of some peroxidase genes induced in biotic stress. The role of both enzyme systems in wheat response/resistance to brown rust is discussed in relation to the oxidative response, the efficiency of resistance, and the presence and origin of particular Lr resistance genes.     

家蚕氟中毒后中肠NADPH-细胞色素P450还原酶和NADPH-细胞色素C还原酶活性的变化

为了探究家蚕对NaF的代谢机制,以家蚕耐氟品种T6和氟化物敏感品种734为材料,从5龄起蚕开始分别添食50、100、200、400 mg/kg NaF溶液处理后的桑叶,检测蚕体中肠微粒体酶液中的黄素蛋白NADPH-细胞色素P450还原酶(CPR)和NADPH-细胞色素C还原酶(CR)的活性变化。氟物化敏感品种734的4个NaF处理组第3天的中肠CPR活性低于对照组,其余时间几乎都高于对照组,400 mg/kg NaF处理组在第4天的CPR活性最高,且各NaF处理组的CPR活性差异显著(P<0.05);耐氟品种T6 NaF处理组和对照组的中肠CPR活性整体上呈先升后降的趋势,几乎都在第2天达到最高值,各组之间的差异不显著(P>0.05)。氟化物敏感品种734的4个NaF处理组的中肠CR活性呈现先升后降的趋势,而对照组呈下降趋势;耐氟品种T6的50、100、400 mg/kg NaF处理组在第1～2天的中肠CR活性呈明显下降趋势,之后的变化相对较小,对照组的CR活性仅在第3～4天略高于NaF处理组,而其余时间NaF处理组的CR活性较高。2个家蚕品种添食不同浓度NaF后的中肠CR活性差异均不显著(P>0.05)。试验结果显示,耐氟品种T6在NaF作用下中肠的CPR和CR活性变化范围(分别为对照组的0.4～2.0倍和0.3～2.9倍)远小于氟化物敏感品种734这2种酶的活性变化范围(分别为对照组的0.6～9.3倍和0.4～4.6倍)。初步推测CPR和CR与家蚕对氟化物代谢具有一定的关联。     

Mechanism of hepatic insulin resistance induced by high-fat diet

AIM: To observed the relationship between oxidative stress and development of insulin resistance in hepatic tissues of Sprague dawley(SD) rats by analyzing reactive oxygen species(ROS) level and NADPH oxidase 3(NOX3) expression in livers. METHODS: Four-week-old male SD rats were fed with high-fat diet containing 20% fat and 20% sucrose for 12 weeks to induce insulin resistance. Plasma insulin level was detected by radioimmunoassay. The content of liver intracellular glycogen was measured using a glycogen assay kit. ROS generation in the liver tissues was assessed by dihydroethidium(DHE) fluorescence. The expression of NOX3 was determined by Western blotting.RESULTS: After 12 weeks of high-fat diet feeding, the content of blood glucose was increased but still maintained in normal level in the rats. However, the index of insulin sensitivity obviously decreased. Hepatic glycogen content in the rats fed with high-fat diet was significantly decreased, indicating that insulin resistance developed. Enhanced ROS production in hepatic tissues of the rats fed with high-fat diet was observed. Importantly, the expression of NOX3 in the liver was up-regulated in response to high-fat diet in vivo.CONCLUSION: High-fat diet feeding decreases insulin sensitivity, enhances ROS level and NOX3 expression, and reduces glycogen content in the livers.     

Regulatory effect of NOX-4 on PI3K signaling pathway in TGF-β1-induced collagenⅠsynthesis from lung cancer cells

AIM: To investigate the regulatory effect of NADPH oxidase-4 (NOX-4) on PI3K signaling pathway in transforming growth factor-β1 (TGF-β1)-induced collagen type I (collagen I)synthesis from lung cancer cells and the mechanisms. METHODS: Human lung cancer A549 cells were cultured in vitro and stimulated with TGF-β1. The expression of NOX family and collagen family at mRNA and protein levels as well as the PI3K class I catalytic subunits and the activation of PI3K signaling pathway was measured. A549 cells were pre-treated with NOX-4 inhibitor diphenyleneiodonium (DPI), and the expression of collagen I at mRNA level as well as the PI3K class I catalytic subunits and the activation of PI3K signaling pathway was measured upon TGF-β1 stimulation. RESULTS: TGF-β1 stimulated the expression of NOX-4 and collagen I at mRNA and protein levels as well as the expression of PIK3CD and the activation of PI3K signaling pathway at a dose-and time-dependent manner. NOX-4 inhibitor DPI partly reversed TGF-β1-induced collagen I expression. Inhibition of NOX-4 down-regulated the degree of TGF-β1-stimulated activation of PI3K signaling pathway without effect on the expression of PIK3CD. CONCLUSION: NOX-4 participates in TGF-β1-induced collagenⅠsynthesis from lung cancer cells via regulating the activation of PI3K signaling pathway. TGF-β1/NOX-4/PI3K signaling pathway axis acts as a regulatory role in collagenⅠsynthesis from lung cancer cells.     

Characterization of reactive oxygen species-involved oxidative damage in Hapalosiphon species crude extract-treated wheat and onion roots

Crude extract-induced oxidative damage using the cyanobacterium, Hapalosiphon sp., was investigated in wheat seedlings ( Triticum aestivum L. cv. Norin 61) and onion seedlings ( Allium cepa L. cv. Raputa II). The analysis of root cell viability or cell death using Evans blue uptake showed that the root-tip cells of wheat and onion lost viability after 24 h and 48 h treatment with 3 g dry weight (DW) L⁻¹ of the crude extract, respectively. Lipid peroxidation was induced in the roots of both species and the shoots of onion, whereas no increase in lipid peroxide formation was observed in the wheat shoots. In onion, the degree of random DNA fragmentation increased with the increasing concentration of the extract and laddering of the DNA was observed with 6 g DW L⁻¹ of the extract, but no apparent DNA ladder formation occurred in the wheat. Pretreatment for 1 h with the NADPH oxidase inhibitors, diphenyleneidonium or imidazole, reduced the crude extract-induced root cell death in both species. From the results, we suggest that the Hapalosiphon sp. crude extract might enhance reactive oxygen species (ROS) production, which causes membrane lipid damage and fragmentation of the DNA of plant cells, resulting in cell death and growth inhibition. The crude extract-mediated phytotoxic damage might be caused by ROS, triggered by NADPH oxidase.     

Four PKC-related proteases involved in atherosclerosis

Atherosclerosis is a multiple-pathogeny disease that consists of many complicated processes, including oxidative stress, endothelium dysfunction, abnormal cell proliferation, apoptosis and extracellular matrix disorder. The pivotal proteases for these courses are NAD(P)H oxidase, eNOS, MAPKs and MMPs, respectively. As an upstream kinase, protein kinase C (PKC) may involve in the development of atherosclerosis through affecting these enzymes.     

镉/铬胁迫对谷子幼苗生长和NADPH氧化酶及抗氧化酶体系的影响

为了探讨重金属镉(Cd)、铬(Cr)胁迫对谷子幼苗生长和生理的影响,采用营养液培养法,检测不同浓度的Cd、Cr胁迫对谷子幼苗生长以及过氧化氢(H_2O_2)含量、叶绿素含量、丙二醛(MDA)含量、NADPH氧化酶和抗氧化酶活性的影响。结果表明:不同浓度(50/1000μmol·L~(-1))两种金属(Cd、Cr)胁迫下,谷子幼苗株高、根长显著减小(P0.05),H_2O_2和MDA含量升高,叶绿素含量降低;随Cd、Cr的浓度升高,根和叶片中NADPH氧化酶活性显著增强,高浓度Cd、Cr可导致过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性显著升高;同时Cd、Cr诱导编码NADPH氧化酶基因Sirboh D和Sirboh F的表达,而超氧化物歧化酶(SOD)活性呈低浓度增加高浓度下降的趋势。由此可知,Cd、Cr对谷子幼苗的胁迫使其发生一系列生理指标变化,造成植株不同程度损伤,植株自身则通过增强抗氧化酶活性对机体起到保护作用。