胡椒4-香豆酸：辅酶A连接酶Pn4CL基因的克隆及表达分析

根据胡椒4-香豆酸：辅酶A连接酶（4-coumarate:coenzyme A ligase, 4CL）基因的部分序列设计引物,运用RACE方法获得其家族成员的1个全长cDNA,命名为Pn4cl,长度2130 bp,开放阅读框1638 bp,编码545个氨基酸。预测Pn4CL分子量为59.57 kDa,理论等电点为5.70。该基因含有AMP-binding（AMP-binding enzyme）、CaiC[Acyl-CoA synthetase (AMP-forming) /AMP-acid ligaseⅡ]、PLN02246、AFD-class I等结合域,具有植物4CL所共有的保守结构域。系统进化分析表明,Pn4CL与北细辛的同源性最高,同时与木兰分支类植物的4CL聚类在一起,与菊分支的进化距离较近,与蔷薇分支的进化距离较远。亚细胞定位表明,该蛋白定位在细胞膜上。Real-time RT-PCR结果表明,该基因受外援激素SA和MeJA诱导表达,同时接种辣椒疫霉菌后,Pn4CL基因的表达量在抗/感2种胡椒中均出现先增加后减少的现象,并且在抗病种质中表达量较高。研究结果为Pn4CL的功能研究提供了理论依据。     

Verrucosamide,a Cytotoxic 1,4-Thiazepane-Containing Thiodepsipeptide from a Marine-Derived Actinomycete

A new cytotoxic thiodepsipeptide, verrucosamide (1), was isolated along with the known, related cyclic peptide thiocoraline, from the extract of a marine-derived actinomycete, a Verrucosispora sp., our strain CNX-026. The new peptide, which is composed of two rare seven-membered 1,4-thiazepane rings, was elucidated by a combination of spectral methods and the absolute configuration was determined by a single X-ray diffraction study. Verrucosamide (1) showed moderate cytotoxicity and selectivity in the NCI 60 cell line bioassay. The most susceptible cell lines were MDA-MB-468 breast carcinoma with an LD₅₀ of 1.26 µM, and COLO 205 colon adenocarcinoma with an LD₅₀ of 1.4 µM. Also isolated along with verrucosamide were three small 3-hydroxy(alkoxy)-quinaldic acid derivatives that appear to be products of the same biosynthetic pathway.     

Sulodexide protects against hyperglycemia-induced retinal vascular injury via suppressing NOX4/NLRP3 pathway

AIM To investigate the effect of sulodexide (SDX) on high glucose-induced damage in retinal microvascular endothelial cells. METHODS (1) High-fat diet combined with intraperitoneal injection of streptozocin were used to induce type 2 diabetes mellitus (DM) followed by injection of saline or SDX in C57BL/6J male mice. Retinal microvascular leakage and density, and the protein levels of NLRP3 inflammasome-related proteins, zonula occludens-1 (ZO-1) and NADPH oxidase 4 (NOX4) were measured. (2) Human retinal microvascular endothelial cells (HRMECs) were treated with normal glucose or high glucose with or without SDX, and were further transfected with siRNA to knock down NOX4, or infected by adenovirus to over-express NOX4. The protein levels of ZO-1, VE-cadherin (VE-Cad), NOX4 and NLRP3 inflammasome-related proteins as well as the level of reactive oxygen species (ROS) were detected. RESULTS Treatment with SDX increased the protein level of ZO-1, attenuated retinal leakage and NLRP3 inflammasome activation, and enhanced the density of microvasculature and the number of ganglion cells in diabetic retinas. The protein levels of ZO-1 and VE-Cad were decreased, while the levels of NOX4, NLRP3 inflammasome-related proteins and ROS generation were increased in high glucose-treated HRMECs. Silencing of NOX4 inhibited high glucose-induced increases in NLRP3 inflammasome and ROS generation, and decreases in the protein levels of ZO-1 and VE-Cad. Over-expression of NOX4 significantly increased the levels of NLRP3 inflammasome-related proteins and ROS generation in HRMECs, and reduced the protein levels of ZO-1 and VE-Cad. Treatment with SDX partly reversed NOX4 over-expression-induced changes. CONCLUSION SDX alleviates hyperglycemia-induced retinal microvascular endothelial injury via inhibiting NOX4/ROS/NLRP3 pathways.     

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.     

Role of IP3R in LH-EGFR-induced mouse oocyte meiotic resumption

AIM: To explore the effect of inositol 1, 4, 5-trisposphate receptor (IP₃R) in luteinizing hormone-epidermal growth factor receptor (LH-EGFR)-induced oocyte meiotic resumption. METHODS: Models of mouse cumulus-oocyte complexs (COCs) culture and follicle culture in vitro were generated to study the effects of 2-aminoethyl diphenyl borate (2-APB) and heparin (IP₃R specific inhibitors) on LH/EGF-induced oocyte meiotic resumption and EGF-induced cumulus cell expansion. Real-time PCR was used to detect the mRNA expression of cumulus expansion-related factors. The changes of the intracellular calcium level were monitored using Fluo 3-AM, and the cGMP level was measured by ELISA. RESULTS: The inhibitors of IP₃R, 2-APB and heparin, dramatically reversed EGF-induced oocyte maturation (P<0.05) and decreased cGMP levels in COCs (P<0.05). In addition, 2-APB and heparin reversed EGF-induced cumulus expansion, and significantly inhibited EGF-induced cumulus expansion-related factor expression (P<0.05). The activation of IP₃R increased intracellular calcium level, and the study found that 2-APB and heparin dramatically reversed EGF-induced elevation of calcium level in cumulus cells (P<0.05). Follicular culture in vitro showed that 2-APB and heparin significantly reversed the LH-induced oocyte maturation (P<0.05). CONCLUSION: LH-EGFR signaling pathway increases calcium level in cumulus cells through IP₃R, resulting in meiotic resumption.     

陕西关中地区猕猴桃溃疡病菌对CuSO4的抗性评价

为明确陕西省猕猴桃细菌性溃疡病菌（Pseudomonas syringae pv.actinidiae,Psa）对CuSO₄的抗性水平及与copA和copB基因表达量的相关性,使用最小抑菌浓度法（MIC）对分离自陕西省关中地区的84株Psa菌株对于CuSO₄的抗性进行检测。在此基础上,使用实时荧光定量PCR方法,对3株不同抗性水平Psa菌株的基因copA和copB与抗铜性的关系进行了分析。结果表明,供试的84株Psa菌株MIC分布在3.75~6.25 mmol·L^-1范围内;不同地区来源的菌株对CuSO₄的抗性表现出明显的差异,其中分离自周至的Psa菌株的抗铜水平明显高于眉县。实时荧光定量PCR分析结果显示,不同浓度CuSO₄条件诱导下,3株不同抗性水平的菌株抗铜基因copA和copB表达水平与CuSO₄浓度成正相关,且菌株的抗性水平越高,基因copA和copB相对表达水平亦越高。陕西省关中地区的Psa菌株对CuSO₄表现出较高的抗性水平,且不同地区的Psa菌株的抗性表现出明显差异;copA和copB基因参与了Psa菌株对铜离子的适应性反应。     

藜麦品系的染色体数目及核型分析

为探明西藏目前主推藜麦的染色体数目及核型,以西藏农牧学院植物科学学院提供的藜麦品系W4为材料,对其进行根尖染色体常规压片法制片,比较采用8-羟基喹啉、秋水仙素和冰冻方法的预处理时间、1mol/L HCl酸解时间对藜麦染色体制片的影响,探讨最优的根尖处理方法并进行核型分析.结果显示:用0.1%秋水仙素溶液(离体)处理3h,1mol/L HCl 60℃酸解13～14 min的总体作用效果最佳;藜麦W4的核型公式为2n=36=32 m(2SAT)+4sm,核型不对称系数为57.87%,核型分类中属2B型.     

关于光呼吸与光合作用关系的研究——Ⅶ、光呼吸是伴随光合作用发生的过程

测定水稻、小麦、棉花等C_3植物和玉米、高粱等C_4植物的光合与光呼吸强度,研究不同处理对光合与光呼吸的影响。植物的光呼吸是与光合作用伴随发生的。强光、高氮等有利光合作用的因素能促进光呼吸;DCMU、叶片失水等抑制光合作用的因素也降低光呼吸。在光合滞后期中,光合强度与光呼吸强度平行上升。在同类植物中,光呼吸强度与光合强度之间有较稳定的比值,C_3、C_4植物的光呼吸占光合分别为30—35％和4％。     

菜籽中4—羟基—3—甲基吲哚硫代葡萄糖苷的离子交换色谱法分离

本文报道用ＤＥＡＥ Ｓｅｐｈａｄｅｘ Ａ－２５作固定相，硫酸钾溶液作流动相的离子交换色谱法，从菜籽中分离４－羟基－３－甲基吲哚硫代葡萄糖苷的方法。