Roles of protein phosphatase 4 in down-regulation of eNOS Ser633 phosphorylation induced by palmitic acid in human umbilical vein endotheli?al cells

AIMTo investigate the roles of protein phosphatase 4 (PP4) in down-regulation of endothelial nitric oxide synthase (eNOS) Ser633 phosphorylation induced by palmitic acid (PA). METHODSHuman umbilical vein endothelial cells (HUVECs) were treated with PA at 25 μmol/L, 50 μmol/L, 100 μmol/L and 200μmol/L for 36 h, or treated with PA at 100 μmol/L for 12 h, 24 h, 36 h and 48 h. Protein phosphatase 2A (PP2A) family inhibitor fostriecin (FST, 20 nmol/L) or okadaic acid (OA, 5 nmol/L) was selected to pretreat the HUVECs for 30 min. Protein phosphatase 4 catalytic subunit (PP4c) siRNA or protein phosphatase 2A catalytic subunit (PP2Ac) siRNA was transfected into the HUVECs. The protein expression levels of of eNOS, PP4c and PP2Ac, as well as the level of eNOS Ser633 phosphorylation, were detected by Western blot. The intracellular nitric oxide (NO) content was measured by DAF-FM DA. RESULTS(1) Compared with control group, the levels of eNOS Ser633 phosphorylation were decreased in PA groups in which the HUVECs were treated with 25 μmol/L, 50 μmol/L, 100 μmol/L and 200 μmol/L PA for 36 h (P<0.05) and 100 μmol/L PA for 24 h, 36 h and 48 h (P<0.05). No significant difference in the level of total eNOS protein expression among all the groups was observed. (2) Compared with control group, both FST and OA pretreatment reversed the reduction of eNOS Ser633 phosphorylation (P<0.05) and the decrease in intracellular NO content (P<0.05) induced by PA. No significant difference in the level of total eNOS protein expression among all the groups was observed. (3) Compared with si-Control group, the PP4c protein expression was significantly reduced (P<0.05), while the level of eNOS Ser633 phosphorylation was significantly increased in si-PP4c group (P<0.05). Although the levels of PP2Ac protein expression declined significantly (P<0.05), the level of eNOS Ser633 phosphorylation remained unchanged in si-PP2Ac group. No significant differencein the level of total eNOS protein expression among all the groups was found. CONCLUSION PA significantly reduces the level of eNOS Ser633 phosphorylation and the content of NO in the HUVECs, which may be due to PA inducing the activation of the PP2A family member PP4 rather than PP2A.     

Effects of dietary caffeine on growth,body composition,somatic indexes,and cerebral distribution of acetyl‐cholinesterase and nitric oxide synthase in gilthead sea bream (Sparus aurata), reared in winter temperature

This study aims at examining the effect of caffeine administration on growth, feed efficiency, and consumption of sea bream (Sparus aurata), reared in winter temperatures. Moreover, it is questioned whether caffeine has a central action in the brain and its effects are partly mediated via central brain mechanisms. For this, we studied the influences of caffeine treatment on the cerebral pattern of the cholinergic neurotransmission and the novel neuromodulator nitric oxide (NO), by means of acetyl‐cholinesterase (AchE) and nitric oxide synthase (NOS) histochemistry. Five different diets containing 0.0, 0.1, 1.0, 2.0 and 5.0 g caffeine kg^?1 of diet were administrated to five groups of fish. Caffeine adversely affected sea‐bream growth at a concentration higher than 1 g kg^?1 diet and increased feed conversion ratio in the treatments of 2 and 5 g kg^?1 (P < 0.05). The daily consumption of feeds was similar to all groups, indicating that caffeine did not influence diet palatability. AChE‐ and NADPH‐diaphorase histochemistry showed densely labeled cells and fibers mainly in dorsal telencephalon, preoptic, pretectal, hypothalamic areas, optic tectum, reticular formation, cerebellum and motor nuclei. When compared with matched caffeine‐treated animals, no differences in the histochemical pattern and cell densities of cerebral AChE and NADPH‐diaphorase were found.     

茶树叶绿素合成相关基因克隆及在白叶1号不同白化阶段的表达

高等植物叶绿素的生物合成对其正常光合作用起关键作用。本文根据前期芯片杂交结果, 采用RT-PCR和RACE技术克隆了3个茶树叶绿素合成相关基因, 分别为谷氨酸-tRNA还原酶(CsGluTR)、叶绿素合酶(CsChlS)、叶绿素酸醋氧化酶(CsCAO), 对应的GenBank的登录号为HQ660371、HQ660370、HQ660369。序列分析表明, CsGluTR基因全长2165 bp, 开放阅读框长1665 bp, 编码554个氨基酸, 推测的蛋白分子量约为60.6 kD, 理论等电点为8.78;CsChlS基因全长1463 bp, 其中开放阅读框长1125 bp, 编码374个氨基酸, 推测的蛋白分子量约为40.5 kD, 理论等电点为8.58;CsCAO基因全长2146 bp, 其中开放阅读框长1611 bp, 编码536个氨基酸, 推测的蛋白分子量约为60.8 kD, 理论等电点为8.03。比对分析表明, 3个基因编码的氨基酸序列与其他植物中同源基因的相似性均在70%以上。利用荧光定量PCR技术检测3个基因在不同白化阶段的表达,表明, CsChlS和CsCAO基因具有明显的表达协同性, 它们在叶片中的表达量与叶片的颜色变化高度同步;而CsGluTR在白化叶片和正常叶片中的表达差异相对较小, 同时在新生芽叶转绿过程中最先恢复正常表达水平。说明在白化叶片中, 叶绿素的合成机制受到较大影响, 叶绿素合成受阻导致的叶片内色素类物质含量降低或消失是叶片白化的直接原因。     

从狼毒大戟中克隆蓖麻烯合成酶基因

本研究采用RACE-PCR技术从狼毒大戟(Euphorbia fischeriana Steud.)根中克隆到一个蓖麻烯合成酶(casbene synthase)基因,命名为EfCS(GenBank登录号为:JN862821)。该基因长1969bp,编码602个氨基酸,具有典型的萜类合成酶的基因结构。对CS基因编码氨基酸序列进行生化特性分析发现其理论等电点为5.36,分子量为69.3648kD。通过疏水性分析,发现CS整体表现为亲水性。进行导肽分析结果发现其具有叶绿体导肽。二级结构预测表明,CS蛋白由α-螺旋、延伸链、β-转角和随意卷曲构成,它们分别占到64.95%、6.64%、3.16%和25.25%。另外,对CS蛋白质进行三级结构预测,发现蓖麻烯合成酶属Ⅰ类萜类合成酶。     

中国野生毛葡萄芪合酶基因表达及对葡萄抗白粉病的影响

[目的]克隆中国野生毛葡萄(Vitis quinquangularis)'丹凤-2'芪合酶(stilbene synthase)基因(STS)并研究其功能,为提高欧洲葡萄(V.vinifera)的白粉病抗性及品质提供依据.[方法]利用同源克隆法获得中国野生毛葡萄'丹凤-2'芪合酶基因VqSTS9和VqSTS21,构建植...     

大肠杆菌BL21(DE3)中表达重组蛋白的研究

为了研究在大肠杆菌BL2 1(DE3)中重组蛋白的表达特点 ,探讨重组蛋白在大肠杆菌中表达时可溶性蛋白与包涵体出现的特点 ,从表达载体中表达了植物ACC合成酶 .经SDS PAGE电泳与双波长扫描分析 ,确定目的蛋白随表达时间、菌液密度的增加而增加 .但是其最佳表达时间为 2 5h ,菌液密度OD6 0 0 为 0 6 ,IPTG的最佳浓度为 0 6mmol L ,此时目的蛋白含量占全菌蛋白含量之比最高 .植物ACC合成酶在大肠杆菌中表达时以包涵体的形式存在 ,未能检测到可溶性蛋白的表达     

柑橘胼胝质合成酶基因家族的表达分析

胼胝质由胼胝质合成酶合成,胼胝质沉积是植物对入侵病原的防御反应。为了探索胼胝质合成酶(Callose synthase,CalS)基因在柑橘中对黄龙病的应答情况,从甜橙基因组中筛选并利用生物信息学分析了柑橘胼胝质合成酶(CsCalS)家族的12个基因。这些基因编码的氨基酸序列与拟南芥中的胼胝质合成酶具有较高的同源性。荧光实时定量PCR结果显示,CsCalS5和CsCalS12在柑橘健康叶片中的表达量高于其他胼胝质合成酶基因。比较黄龙病感病材料和健康对照材料,发现CsCalS5、CsCalS7和CsCalS8在感病的‘砂糖橘’和‘强德勒’柚中均上调表达,可能参与柑橘对黄龙病病原的应答反应。     

干旱胁迫对不同品系藜麦内黄酮和抗氧化性的影响

利用不同浓度的PEG溶液模拟干旱胁迫,分析了干旱胁迫下不同品系藜麦叶片中黄酮类化合物合成上游的苯丙氨酸解氨酶(PAL)、肉桂酸4-羟基化酶(C4H)及4-香豆酸辅酶A连接酶(4CL)此3个关键酶的活性以及种子内黄酮含量和清除DPPH·能力,探究干旱胁迫对不同藜麦种子内黄酮合成以及体外抗氧化活性的影响。结果表明:(1)随着干旱胁迫增加,三种酶活性均成先增后降趋势,但不同酶之间存在差异,PAL和4CL在5%PEG胁迫下达到最大,藜麦品系NK1、NK2和NK5的C4H活性在10%PEG时达到最大,但NK3和NK4在5%PEG胁迫下达到最大;在CK和5%PEG胁迫下藜麦品系NK3的3种酶活性较高,但在10%PEG和15%PEG胁迫下酶活性迅速降低。(2)随着干旱胁迫加剧,各品系藜麦种子内黄酮含量呈先上升后下降的趋势,在CK和5%PEG胁迫下,藜麦品系NK3种子黄酮含量最高分别为2.94 mg/g和3.61 mg/g,在10%PEG和15%PEG胁迫下,NK5种子黄酮含量最高分别是2.92 mg/g和2.66 mg/g。(3)抗氧化试验表明,各品系藜麦种子内黄酮对DPPH·的清除率在5%PEG胁迫处理时达到最大,NK1、NK2、NK3、NK4和NK5清除率分别是88.52%、87.65%、90.36%、86.53%和89.38%,其中NK3 IC50(半抑制浓度)最大为9.871μg/mL。本研究结果为理解藜麦体内黄酮合成体系和抗氧化剂资源的筛选提供理论依据。     

白木香倍半萜合酶基因AsVS的克隆与表达分析

法尼基焦磷酸(farnesyl diphosphate, FPP)是植物萜类化合物合成途径的重要前体之一,经不同的酶催化可形成各类萜类化合物,Vetispiradiene合酶可催化FPP形成Solavetivone和Lubimin的前体Vetispiradiene。本研究利用白木香转录组数据,首次克隆了编码白木香Vetispiradiene合酶的基因(命名为As VS, Genbank登录号为MH378283),AsVS基因序列全长为1 632 bp,编码543个氨基酸,该氨基酸序列与马来沉香倍半萜合酶聚类于同一分支,具有较近的亲缘关系,该蛋白植物倍半萜的保守性结构域DDXXD,NST/DTE和R(R)X8W,相对分子量为62.73 kD,理论等电点为5.51,包含40个磷酸化位点;二级结构以α螺旋和无规则卷曲为主;不含跨膜结构域。RT-qPCR分析结果表明AsVS基因在结香剂处理过程中于白木香茎干样品中持续上调表达,其第6天及第9天的表达量分别为对照处理的2.36和5.02倍。此结果为进一步研究As VS基因在白木香萜类化合物的合成及沉香致香成分富集中的功能提供了理论基础。     

谷氨酸棒杆菌麦芽寡糖基海藻糖合成酶基因的克隆与表达

采用PCR方法从谷氨酸棒杆菌（Corynebacterium glutamicum）基因组中分离得到2.4 kb的麦芽寡糖基