Role of ghrelin in cell protection by up-regulating HSP70 through ERK1/2 signaling pathway in PC12 cells

AIM:To study the role of ghrelin in cell protection by up-regulating heat shock protein 70 (HSP70) and inhibiting apoptosis induced by oxidative stress through extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathway in the PC12 cells. METHODS:Sodium nitoprusside (SNP) was used to induce oxidative stress injury in the PC12 cells. The cultured PC12 cells were divided into SNP-injured group (incubated with SNP at 0.5 mmol/L for 6, 12, 18 and 24 h), ghrelin pretreatment group (ghrelin at 100 nmol/L was given 30 min before adding SNP); HSP70 inhibitor group (quercetin at 10 μmol/L was added 60 min before ghrelin treatment), ERK inhibitor group (ERK 1/2 inhibitor PD98059 was added 60 min before ghrelin treatment) and control group (added same amount of culture medium only). The apoptotic rate was detected by flow cytometry. The protein expression was determined by Western blot and immunocytochemistry. RESULTS:Compared with control group, the apoptotic rate of PC12 cells in SNP-injured group was significantly increased (P<0.05). Compared with SNP-injured group, ghrelin (100 nmol/L) pretreatment significantly inhibited SNP-induced apoptosis of PC12 cells (P<0.05), and significantly up-regulated the protein expression of HSP70 (P<0.05). Time-effect analysis showed that ghrelin had the most significant effect at 18 h after SNP injury. Quercetin, an inhibitor of HSP 70, significantly reduced the anti-apoptotic effect of ghrelin (P<0.05). Ghrelin pretreatment promoted the phosphorylation of ERK1/2. ERK1/2 inhibitor PD98059 significantly inhibited the effects of ghrelin on up-regulation of HSP70 expression (P<0.05). CONCLUSION:Ghrelin upregulates the expression of HSP70 and inhibits the apoptosis in the PC12 cells induced by oxidative stress by promoting the phosphorylation of ERK1/2.     

豫北山区“粮—草—羊”高效平衡养羊模式现状

从粮食作物、经济作物及饲草的种植,饲草料的加工调制与利用,全舍饲肉羊的养殖及杂交繁育,以及羊粪尿的资源化利用等方面介绍了豫北山区“粮—草—羊”高效平衡养羊模式发展现状,以期为促进豫北山区资源可持续利用、山区生态环境良性循环和社会经济可持续发展提供思路。     

大豆植株分杈数自动提取算法研究

根据大豆植株分杈个数可以预估大豆的产量。为了满足现代农业预测农产品产量的需要,本文提出一种大豆植株分杈数自动提取算法,即首先将采集到的大豆植株图片进行灰度变换、滤波、阈值分割得到二值图像,再进行形态学中的闭运算操作得到大豆植株的轮廓,然后基于Zhang并行快速细化算法提取骨架,最后借助改进的角点检测算法对大豆植株的所有分杈点进行标记并自动统计分杈数,仿真结果与大豆植株实际分杈点一致。     

三种融合标签对大熊猫轮状病毒结构蛋白VP6-VP7可溶性表达的影响

大熊猫轮状病毒(giant panda rotavirus,GPRV)是引起幼龄大熊猫腹泻的主要病原,对圈养大熊猫产生了较大的危害。轮状病毒结构蛋白VP6是一种载体蛋白,可介导黏膜免疫反应。VP7是轮状病毒结构蛋白中主要的中和抗原。因此,VP6-VP7的融合表达作为候选抗原对该病的防治具有重要的意义。传统大肠埃希菌原核表达存在表达量低、可溶性差以及纯度低等弊端。本研究使用醛缩酶(EDA)、谷胱甘肽S-转移酶(GST)、麦芽糖结合蛋白(MBP)3种融合标签,以实现获得表达量高和纯度高的GPRV-VP6-VP7重组表达蛋白。将扩增的VP6、VP7基因片段利用同源重组酶构建到含3种融合标签的表达载体pET21b上,将重组质粒转化至大肠埃希菌Rosetta(DE3)感受态细胞中进行低温诱导表达。用Ni-柱亲和层析法纯化目的蛋白,SDS-PAGE和Image J分析蛋白表达量和可溶性,Western blot分析得到表达的重组表达蛋白正确且具有蛋白活性。实验结果证明,EDA标签能显著促进VP6-VP7蛋白的原核可溶性表达,提高VP6-VP7蛋白表达量。     

PPARγ mediates effects of diosgenin on proliferation and apoptosis in human glioblastoma U87MG cells

AIM:To investigate the effect of diosgenin (Dio) on the proliferation, apoptosis and expression of peroxisome proliferator-activated receptor γ (PPARγ) in human glioblastoma U87MG cells and its possible mechanism. METHODS:Human astrocytes (HA) and U87MG cells were cultured in vitro and treated with Dio (0, 10, 20, 30, 40 and 50 μmol/L) and GW9662 (5 μmol/L) for 48 h, and then the cell viability was detected by CCK-8 assay. Cell colony formation assay was used to assess the proliferation potential. Flow cytometry was used to analyze the cell cycle distribution and apoptosis. The mRNA expression level of PPARγ was measured by RT-PCR. Western blot was used to determine the protein levels of PPARγ, cyclin D1, cyclin E1, Bcl-2 and Bax. RESULTS:Dio had no significant influence on the viabi-lity of HA (P>0.05). However, Dio remarkably reduced the viability of U87MG cells in a dose-dependent manner (P<0.05) with IC₅₀ of 24.31 μmol/L. Meanwhile, Dio remarkably diminished colony formation ability (P<0.05), induced G₀/G₁ phase arrest of the cell cycle and apoptosis (P<0.05), up-regulated the expression of PPARγ at mRNA and protein levels, increased the protein level of Bax (P<0.05), and down-regulated the protein levels of cyclin D1, cyclin E1 and Bcl-2 (P<0.05) in a dose-dependent manner. However, these effects induced by Dio were inhibited by GW9662 (P<0.05), a specific inhibitor of PPARγ. CONCLUSION:Dio may inhibit proliferation and induce apoptosis in human glioblastoma U87MG cells most likely via up-regulating the expression of PPARγ, and then down-regulating the protein levels of cyclin D1, cyclin E1 and Bcl-2, and up-regulating the protein level of Bax.     

伐地那非增加EGCG-β-乳球蛋白纳米粒对肝癌细胞的抑制作用

较高浓度的EGCG才能抑制癌细胞的增殖,通过纳米化和EGCG与其他药物的联合使用是提高EGCG生物活性的重要策略。本研究将EGCG和伐地那非（VD）同时包埋于β-乳球蛋白（β-Lg）纳米载体中,制备出EGCG-VD-β-Lg纳米粒（EVβ-NPs）,体外试验证实,EVβ-NPs能提高人肝癌细胞（HepG2细胞）中Caspase-3活性,使HepG2细胞在S期产生明显的阻滞,诱发细胞核分裂,从而导致HepG2细胞凋亡。研究结果表明,将EGCG与微量的VD联合使用,并通过纳米化包埋可以显著提高EGCG的抗癌活性。这一方法在EGCG抗癌制品的开发方面具有潜在的价值。     

基于多反应监测质谱技术的水稻叶片蛋白绝对定量方法

【目的】建立水稻叶片蛋白的多反应监测(MRM)质谱绝对定量方法。【方法】水稻叶片蛋白经含1.0%十二烷基硫酸钠(SDS)的磷酸盐(PBS)缓冲液提取,丙酮沉淀除杂纯化、胰蛋白酶消化,酶解液经液相色谱分离,MRM质谱监测,外标法定量。【结果】向提取缓冲液中加入1.0%SDS可增强水稻叶片中16种靶蛋白和总蛋白质的提取效果;不同有机试剂处理,总蛋白质沉淀量存在显著差异(P<0.05),沉淀能力从强到弱依次为乙腈>丙酮>异丙醇>甲醇>乙醇;对于16种目标蛋白,总体以丙酮沉淀效果最好,其次是异丙醇和乙腈,甲醇和乙醇效果较差。该方法线性范围均达到3个数量级,定量限为0.1~2.5 nmol/L,灌浆期16种水稻叶片蛋白质含量为6.0~2818.1μg/g,相对标准偏差均小于14%。【结论】SDS可显著提高水稻叶片蛋白提取效果,采用丙酮或乙腈可获得较好的蛋白沉淀效果,但不同蛋白质略有差异。结合MRM质谱监测技术可实现水稻叶片蛋白的绝对定量,方法线性范围宽、敏度高、重复性好。     

色氨酰-tRNA合成酶基因WRS1调控水稻种子发育

【目的】氨酰-tRNA合成酶（aminoacyl-tRNA synthetases, aaRSs）与遗传信息传递密切相关,已发现植物中aaRSs家族蛋白在维持翻译功能之余,还参与配子发生与胚发育、质体的早期发育以及免疫信号的感知与病害防御等生物学过程。本研究利用水稻胚乳发育缺陷突变体,分析水稻色氨酰-tRNA合成酶（WRS1）在胚乳发育中的作用,证明WRS1基因编码一个影响水稻胚乳发育的关键因子。【方法】本研究通过甲烷磺酸乙酯（ethyl methane sulfonate, EMS）诱变籼稻（Oryza sativa subsp. indica）品种N22,筛选到一个稳定遗传的水稻粉质胚乳突变体（wrs1）,图位克隆获得目标基因。对wrs1成熟种子进行形态学观察以及淀粉相关理化性质测定,利用细胞学切片分析wrs1发育中胚乳的结构,利用实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）和GUS活性染色分析基因表达模式,通过qRT-PCR比较野生型与突变体花后12 d胚乳中淀粉合成相关基因表达情况,免疫印迹检测野生型与突变体成熟种子中淀粉合成酶蛋白积累情况,使用全自动氨基酸分析仪测定游离氨基酸含量。【结果】 wrs1突变体幼苗表现出明显的发育滞后且逐渐蔫萎死亡,从杂合突变体（WRS1wrs1）中分离到的粉质籽粒呈现明显的腹部皱缩,粒厚、千粒重下降,同时总淀粉含量下降,糊化淀粉的峰值黏度和崩解值均低于野生型。wrs1突变体发育胚乳中复合淀粉颗粒变小,排列疏松。WRS1定位于第12染色体长臂约183 kb的区间内,测序发现编码色氨酰-tRNA合成酶（tryptophanyl-tRNA synthetase, WRS）基因的第6外显子上发生单碱基替换,导致一个保守位置上的甲硫氨酸被替换。wrs1突变体中大部分淀粉合成相关基因表达量下调,且野生型与突变体间基因表达的变化与相应蛋白积累的差异存在不一致的趋势。wrs1突变体籽粒中蛋白质积累降低,而游离氨基酸含量显著升高。【结论】 WRS1编码色氨酰-tRNA合成酶,该基因突变后通过影响氨基酸稳态和蛋白质合成,造成淀粉合成相关基因异常表达从而影响淀粉的合成与积累,导致种子发育缺陷。     

玉米热激蛋白ZmHSP70-12基因的克隆及表达分析

热激蛋白70(HSP70)是原核和真核细胞中普遍存在的一种高度保守的分子伴侣。从玉米中克隆1个HSP70家族成员。该基因cDNA序列全长为1 992 bp,开放阅读框为2 352 bp,编码663个氨基酸,蛋白质分子量约75.0 kD。蛋白结构预测及同源比对分析表明,该基因编码蛋白含ATPase位点和HSP70保守结构域,与拟南芥AtHSP70-12序列高度相似,命名为ZmHSP70-12。蛋白亚细胞定位显示,ZmHSP70-12蛋白在内质网中表达。实时荧光定量PCR分析表明,ZmHSP70-12对非生物胁迫高温、干旱均具有明显的应答反应,推测ZmHSP70-12是玉米中与胁迫逆境相关的基因。     

接种根瘤菌对西南地区大豆光合性能和固氮能力的影响

为探讨接种根瘤菌对西南地区大豆固氮能力的有效性，在大田试验条件下，以根瘤菌株 Bradyrhizobium japonicum5038和B. japonicum 5136侵染南黑豆20和南夏豆25，比较两个大豆品种接种后植株的光合特性和叶绿素 含量，考察根瘤数量并测定分析其蔗糖含量、豆血红蛋白含量和固氮酶活性。结果表明：接种后两大豆品种叶片 chla/b值显著下降，叶绿素含量、净光合速率和气孔导度显著增加；根瘤数、单株瘤重、植株地上部和地下部生物量 均显著增加，单个瘤重和根冠比则无显著变化；根瘤内蔗糖含量和豆血红蛋白含量均显著增加，固氮酶活性提高。 不同大豆品种对不同菌株的响应不同，南夏豆25与根瘤菌B.japonicum5136的适配性最好，接种后大豆光合性能和 固氮能力的改善效果明显优于南黑豆20。