木薯金属硫蛋白基因的克隆和表达分析

金属硫蛋白(metallothionein,MT)分子量小,半胱氨酸含量高,具有消除离子自由基,减弱重金属毒性和减少体内活性氧积累等功能。为探究木薯金属硫蛋白基因(MeMT)的原核表达能力和对活性氧的耐受性,本研究通过木薯cDNA克隆获取MeMT并将其连接至pET-28a,在BL21(DE3)菌株中成功诱导其表达。H2O2耐受性分析发现,与空载菌株相比,表达MeMT蛋白的DE3菌株对H2O2的耐受水平较高。RT-qPCR技术检测显示,在H2O2胁迫下,3 h时木薯叶片中MeMT的表达水平明显升高,但6 h时恢复甚至略低于对照组,证明MeMT在木薯中参与ROS应答过程。该结果对研究木薯抗逆性和产量的提高具有重要意义。     

铜、锌离子影响丹参金属硫蛋白MT2基因表达

植物金属硫蛋白在金属离子的储存、运输和代谢及降低重金属离子的毒性等方面起着重要作用本文研究了Cu2 、Zn2 对丹参毛状根中金属硫蛋白基因表达的影响.以6,7-V为液体培养基,分别以缺乏正常及原培养基100、500、1 000倍的cu2 、Zn2 处理,采用半定量PCR法检测不同时间点时金属硫蛋基因的表达情况.结果发现Cu2 、Zn2 诱导金属硫蛋基因的表达量分别在100倍处理量时的9 h和6 d达到峰值Cu2 、Zn2 能显著诱导丹参金属硫蛋白基因的表达,该基因可能在丹参对铜、锌元素的吸收方面发挥着重要的作用.     

外源性金属硫蛋白对奶牛泌乳性能的影响

选取24头经产泌乳中国荷斯坦牛,随机等分为Ⅰ、Ⅱ、Ⅲ、Ⅳ 4组,分别每头静脉注射经生理盐水溶解的Zn-MT 0（对照）、5.0、10.0和15.0mg,以探讨外源性金属硫蛋白（MT）对奶牛泌乳性能的影响。结果表明,Ⅱ组、Ⅲ组和IV组的4%标准乳产量均显著（P〈0.05）高于对照组;各试验组奶中MT含量显著（P〈0.05或P〈0.01）高于对照组;各组间的乳脂率虽无显著差异（P〉0.05）,但也呈现出试验组高于对照组的趋势。说明MT是一种能够有效调控奶牛泌乳性能的生理活性物质和高效抗氧化剂。     

镉中毒解毒机理的研究：硒化合物的预防和治疗作用初探

对大鼠组织胞液(Cytosol)的体外试验结果表明:亚硒酸钠、硒酸钠及硒化钠以明显影响[~(109)Cd]标记的氯化镉在组织胞液中的分布。硒酸钠、亚硒酸钠有较强的封闭金属硫蛋白(MT)与镉结合部位、激活分子量较大的蛋白质,捕获胞液中[~(109)Cd]的作用,还可使已经和MT结合的[~(109)Cd]被剥离,随后,[~(109)Cd]转入分子量较大的蛋白质上;亚硒酸钠还可使[~(109)Cd]转入分子量为32000～35000的蛋白质上。硒化钠仅有较弱的剥离与MT结合的镉,使之转入分子量较大的蛋白质上,但几乎不能阻止镉与MT结合。镉与大、中分子量蛋白质结合力远较它与MT的结合力弱,因而以能有利于镉被排出体外。     

Metallothionein expression in benign and malignant canine mammary gland tumours

The presence of metallothioneins (MTs) were demonstrated immunohistochemically using a monoclonal antibody (E9) against a conserved epitope of I and II isoforms in canine mammary tumours. In a semiquantitative analysis MT expression in the tumour cells was observed in 54/54 cases of benign and 32/40 malignant mammary neoplasms. A statistically significant difference at the level of P<0.01 was observed for MT expression between benign and malign mammary tumours in terms of immunoreactivity score. It is concluded that immunohistochemically demonstrated MT expression is significantly associated with benign canine mammary tumours.     

Protective mechanism of metallothionein on cultivated rat cardiomyocytes in hypoxic preconditioning

AIM: Studying the mechanism of protective role of metallothionein(MT) in hypoxic preconditioning(HPC) of cultivated rat cardiomyocytes. METHODS:Using the model of hypoxia/reoxygenation of cultivated rat cardiomyocytes. Determining the contents of MT, malonyldialdehyde (MDA)-metabolism product of lipid peroxidation and the activities of Na⁺-K⁺ATPase, Ca²⁺-Mg²⁺ATPase of cardiomyocytes 24h after HPC, also determining the relevant changes after using MT antibody. RESULTS: After 24 h in HPC, the contents of MT and activities of Na⁺-K⁺ATPase, Ca²⁺-Mg²⁺ATPase were obviously higher than those in the control and hypoxia/reoxygenation(P＜0.05), and the contents of MDA were decreased remarkedly (P＜0.01). Then after using MT antibody, the activities of two enzyme were progressively decreased and the contents of MDA were significantly higher than those in the control and MT antibody-free groups(P＜0.01). CONCLUSION: HPC may induce excessive synthesis of MT, and MT can protect myocardial reoxygenation injury by eliminating lipid peroxidation and rising the activities of Na⁺-K⁺ATPase and Ca²⁺-Mg²⁺ATPase.     

Metallothionein inhibits homocysteine-induced proliferation of rat vascular smooth muscle cells

AIM:To investigate the effect of metallothionein(MT) on proliferation of rat vascular smooth muscle cells (VSMCs) stimulated by homocysteine and its mechanism. METHODS:VSMCs proliferation was measured by [³-H]-TdR incorporation, mitogen-activated protein kinase(MAPK)activity were determined by immunoprecipitation method, the intracellular contents of MT and malondialdehyde (MDA)were assayed by -hemoglobin saturation method and TBA reaction, respectively, and lactate dehydrogenase (LDH) leakage was measured by NADH oxidation. RESULTS:Hcy(10^-6-10^-4 mmol/L) stimulated [³-H]-TdR incorporation by the VSMCs in a concentration-dependent manner. Compared with control, [³-H]-TdR incorporation in VSMCs treated with 0.1 mmol/L Hcy was increased by 4.2 fold (P＜0.01). Meanwhile, Hcy enhanced MAPK activity, MDA formation and LDH release (P＜0.01)in a concentration-dependent manner. Treatment of VSMCs with MT alone did not change above parameters, compared with control. However, MT (10^-6-10^-4 mol/L)attenuated significantly Hcy-stimulated proliferation of VSMCs (P＜0.01)in a concentration-dependent manner. And MT inhibited obviously Hcy-induced activation of MAPK activity, MDA formation and LDH release. Preincubation of VSMCs with 0.5 mmol/L ZnCl₂ for 6 h induced an increase cellular MT content by 5.7-fold (P＜0.01). The MT-overexpressed VSMCs resisted Hcy-stimulating action on MAPK activity, MDA formation and LDH leakage (P＜0.01). CONCLUSION:These results show that MT has an inhibitory effect on Hcy-induced VSMCs proliferation, and that MT could inhibit Hcy-stimulated MAPK activity and lipid peroxidation.     

光敏生物素标记IgG检测农产品重金属污染

重金属污染的植物体内会产生较高的金属硫蛋白(MT),实验以光敏生物素标记用MT免疫家兔产生的抗体,用碱性磷酸酶标记亲和素,对MT进行免疫印迹检测,方法简单准确,标记的IgG最低检出量为1 ng/mL。通过对天水秦城区不同地域采集到的16种农产品样本进行检测,其检测结果与样本中金属硫蛋白的含量测定结果一致。     

真核细胞内的铜转运及平衡机制研究进展

以果蝇为例,阐述了铜在动物体内的摄入、分配、排泄以及转录水平调控过程。果蝇细胞对铜的摄取主要通过金属反应转录因子MTF调节转运蛋白Ctr与金属硫蛋白mt之间的表达调控铜离子的摄取。进入胞浆内的铜主要通过ATX1、CCS和Cox17这3种铜伴侣蛋白经3条不同途径进行转运和调节。金属硫蛋白与P型ATP7酶在铜的排泄中起重要作用。     

柽柳金属硫蛋白基因的克隆及分析

应用生物信息学技术从柽柳cDNA文库中分离出了一种金属硫蛋白全长cDNA序列,去除PolyA后该基因全长517bp。其中,5′非翻译区31bp,3′非翻译区267bp,开放读码框(ORF)长219bp,编码72个氨基酸,含13个半胱氨酸Cys,分布在蛋白质的N端和C端,呈CXC形式排列,基因编码蛋白的分子量为7.1kD,等电点为4.71。疏水性分析表明,蛋白的30～50个氨基酸之间有较强的疏水性,经过Pfam15.0(St.Louis(USA))分析证明,为金属硫蛋白家族基因。金属硫蛋白基因参与了植物的发育、胚胎发生、抵抗逆境胁迫等多种生理过程,是一种重要的功能基因。此基因的Genbank登录号为AY620987(基因),AAT39518(蛋白)。