Metallothionein protects rat hepatic nuclear nucleoside triphosphatase from hydroxyl radical-induced suppression

AIM:Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotection during metal exposure and oxidative stress. The present study was designed to investigate whether MT can directly protect NTPase on nuclear envelope from damage induced by hydroxyl radical.METHODS:Isolated hepatic nuclei from rat liver were exposed to Fe²⁺/H₂O₂ with or without MT, and the NTPase activity on nuclei was assayed using ATP and GTP as substrate, respectively.RESULTS:Incubation of rat hepatic nuclei with the Fe²⁺/H₂O₂ (in μmol·L^-1/μmol·L^-1 : 0.1/0.5, 0.5/2.5, 1/5, 5/25) resulted in a concentration-dependent decrease in nuclear NTPase activities (P＜0.01). Incubation of hepatic nuclei with different concentrations of MT (10^-9-10^-4mol·L^-1)and Fe²⁺/H₂O₂ (1 μmol·L^-1/5 μmol·L^-1) for 10 min, nuclear NTPase activities were increased in a MT concentration-dependent fashion as compared with that of incubation with Fe²⁺/H₂O₂(1 μmol·L^-1/5 μmol·L^-1) alone. When MT was at 10^-4 mol·L^-1, TNPase activities reversed to (102±10) nmol·mg^-1 protein·min^-1(for ATP as substrated) and (131±12) μmol·g^-1 protein·min^-1(for GTP as substrate), which had no significant defferences from that of the controls (112±8 and 142±10 μmol·g^-1 protein·min^-1, respectively) (P＞0.05). In addition, incubation of hepatic nuclei with only MT had no effect on nuclear NTPase activity. CONCLUSION:These data demonstrate that hydroxyl radical generated from Fe²⁺/H₂O₂ might attack nuclear NTPase. MT antagonistically reduces toxicity of Fe²⁺/H₂O₂ system to the NTPase.     

印染污水对银鲫卵黄蛋白原和金属硫蛋白水平的影响

分析了不同浓度印染污水对银鲫(Carassius auratus)的生殖相关指标和金属硫蛋白(MT)的影响.设计1个对照组和2个试验组,对照组采用天然的水库水,试验组分别用浓度为0.1％和0.2％的印染污水养殖银鲫,每组3个平行,每个平行随机放入50尾银鲫,试验期为4周.结果表明,养殖4周后,0.1％印染污水组银鲫的肝体指数和血清卵黄蛋白原(VTG)水平显著上升(P＜0.05),0.2％印染污水组银鲫的血清VTG水平显著低于对照组(P＜0.05),印染污水对银鲫性腺指数没有影响.0.1％印染污水组银鲫在第2、3、4周,肝脏MT水平均显著高于对照组(P＜0.05),0.2％印染污水组银鲫肝脏MT水平在第3周时显著升高(P＜0.05),印染污水对银鲫肾脏MT水平没有影响(P＞0.05);表明印染污水能够影响银鲫繁殖功能和组织MT的水平.     

水稻新型MT 基因的鉴定及其基因家族分子特征分析

[目的]探讨水稻MT基因家族各成员在基因结构上的差异及相互间的进化关系。[方法]从经重金属处理的水稻幼苗中分离到3个新的植物MT,对其进行RT-PcR和Northem杂交分析以及水稻和拟南芥MT基因家族的序列比对和进化分析。[结果]RT-PCR和Northern杂交分析表明,水稻中3个MT基因的表达均受重金属的诱导,在幼苗根部进行特异性表达。由水稻和拟南芥biT基因家族的序列比对提出植物MT的新分类方式。水稻I类OsMT基因家族的所有10个成员,定位到6条染色体上。同一类型的OsMT有高度的氨基酸序列保守性,相似的Cys排列方式。新发现的3个OsMT家族在单子叶植物水稻中独有,属4型MT。[结论]水稻和拟南芥biT基因家族进化分析显示,植物MT至少发生在种子植物出现前,而4型MT可能发生于单子叶植物和双子叶植物分离后。     

酵母耐铜基因CUP1改良荧光假单胞菌耐铜性研究初报

利用根际促生菌（PGPR）荧光假单胞菌（Pseudomonas fluorescens）等微生物的生物修复作用可治理土壤重金属污染。酿酒酵母耐铜基因CUP1编码铜金属硫蛋白（Cu-MT）,对铜有很高的亲和性,可拮抗重金属铜毒性。为提高荧光假单胞菌的生物修复能力,将酿酒酵母耐铜基因CUP1克隆至荧光假单胞菌。通过引物设计,将SD序列添加至酵母耐铜基因CUP1编码序列前端,PCR扩增得到约200bp的CUP1片段,克隆至测序载体pUC19,经测序证明正确后,胶回收CUP1片段连接到具有广泛宿主范围的柯斯质粒pLAFR3上,经三亲接合转移至荧光假单胞菌AS1.1381,得到重组菌株AS1.1381/pL3CUP1。铜实验表明,重组菌株AS1.1381/pL3CUP1的抗铜能力（6mM Cu2+）显著高于AS1.1381/pLAFR3（4.5mM Cu2+）,重组菌株抗铜性得到提高     

Metallothionein involvement in myocardial protection of basic fibroblast growth factor

AIM: To observe whether metallothionein plays a role in cardiac protective effect of basic fibroblast growth factor on anoxic/reperfusion (A/R) injury in cultured cardiomyocytes and study the possible mechanism of cardiac protection by bFGF. METHODS: The present study made the model of myocyte A/R injury after having a 24 h incubation by bFGF (10^-10, 10^-9 and 10^-8 mol/L) and bFGF (10^-9 mol/L) +PD₀₉₈₀₅₉, respectively. We measured the levels of MT and MDA in myocytes, and the changes of LDH and protein in cultured medium. We also counted the number of viable cell in groups. RESULTS: The contents of myocardial MT were significantly increased after treatment by bFGF. The levels of MT in 10^-10 mol/L, 10^-9 mol/L and 10^-8 mol/L bFGF treated groups increased 54%, 62% and76%, respectively, compared with the A/R group, and the number of viable cell were also greatly increased, LDH and protein leakage in cultured medium and MDA contents in myocyte were dramatically decreased in bFGF treated groups. All the protection were completely disappeared with the inhibition of MT production with PD₀₉₈₀₅₉, the inhibitor of mitogen-activated protein kinase (MAPK). CONCLUSION: MT involves in the protection of bFGF in cultured cardiomyocytes. It might be related with activation of MAPKase.     

兔肝金属硫蛋白提取液的稳定性

研究Tris-HCl浓度、pH、温度、光照和乙醇对金属硫蛋白提取液稳定性的影响,为金属硫蛋白的研究提供资料.结果表明,金属硫蛋白在低浓度Tris-HCl中较稳定,0.01和0.02 mol/L Tris-HCl对其稳定性影响最小;pH对金属硫蛋白稳定性影响较大,pH 6～7时较稳定;光照对金属硫蛋白稳定性影响比较明显,其对硫蛋白具有破坏作用;乙醇对金属硫蛋白有明显的保护作用;金属硫蛋白具有很强的耐热性.     

镉诱导黄瓜金属硫蛋白的分离、纯化和鉴定

[目的]通过不同浓度Cd2+诱导黄瓜产生金属硫蛋白,探寻植物金属硫蛋白含量与Cd2+浓度关系。[方法]用不同浓度Cd2+诱导黄瓜幼叶组织产生金属硫蛋白,经Sephadex G-50凝胶过滤层析纯化后用SDS-聚丙烯酰胺凝胶电泳测定黄瓜金属硫蛋白的分子量。[结果]1 000 mg/LCd2+诱导黄瓜幼叶产生金属硫蛋白的产量最高,可达2.62μg/g。黄瓜金属硫蛋白的分子量为9.5 kD。[结论]黄瓜是一种对Cd2+敏感的植物。Cd2+诱导的金属硫蛋白含量与Cd2+浓度呈正相关。     

柽柳、星星草金属硫蛋白基因双价植物表达载体的构建及其在烟草中的表达

将在柽柳(Tamarix and rossowii)和星星草(Puccinellia tenuiflora)cDNA文库中克隆到的金属硫蛋白基因PCR扩增后,连接到载体pROKII上,构建了分别由2×CaMV35S启动子调控的柽柳金属硫蛋白基因和星星草金属硫蛋白基因双价植物表达载体pRCXM,经PCR检测及酶切鉴定证实pRCXM构建成功。通过农杆菌介导的叶盘法转化烟草(Nicotiana tobacum Longjiang911),获得了抗卡那霉素的转基因植株。经初步检测结果显示,外源基因已整合进烟草基因组。重金属抗性结果证明:转基因烟草可以在CdCl2的浓度为300μmol/L的培养基中生长,而对照烟草只能在CdCl2的浓度为100μmol/L的培养基中生长。     

诱导前后家兔组织中金属硫蛋白含量的变化

采取皮下注射硫酸锌,诱导家兔组织产生锌-金属硫蛋白(Zn-MT)。结果:诱导期间肝组织中Zn-MT逐步升高,后期增幅减慢,最后高达5.38mg/g湿组织重;诱导后肾、血、肺和肠中Zn-MT的含量也有较大升高,兔肉中Zn-MT升高幅度最小且含量最低,为0.14mg/g湿组织重。实验表明:除了兔肝以外,肾、血、肺和小肠等组织也可用作Zn-MT提取和纯化的原料。     

根癌农杆菌介导的mMT-IcDNA转化枸杞及其表达的研究

报道了小鼠金属硫蛋白－ＩｃＤＮＡ通过根癌农杆菌介导转入枸杞,并在枸杞体内表达产生富集锌的转基因枸杞。用带有嵌合ｍＭＴ－ＩｃＤＮＡ及ｃａＭＶ３５ｓ启动子的含非致癌性Ｔｉ质粒载体的概癌农杆菌ＬＢＡ４４０４（ｃａＭＶ３５ｓ：ｍＭＴ－ＩｃＤＮＡ：ｎｏｓ）感染枸杞幼茎外植体,在含有Ｋｍ１００μｇ／ｍｌ和Ｚｎ＾２＋５０μｍｏｌ／Ｌ的选择培养基上筛选转化的愈伤组织,并在含Ｋｍ５０μｇ／ｍｌ,Ｚｎ＾２＋１００μｍ