Increased demyelination and axonal damage in metallothionein I+II-deficient mice during experimental autoimmune encephalomyelitis

Metallothioneins I+II (MT-I+II) are antioxidant, neuroprotective factors. We previously showed that MT-I+II deficiency during experimental autoimmune encephalomyelitis (EAE) leads to increased disease incidence and clinical symptoms. Moreover, the inflammatory response of macrophages and T cells, oxidative stress, and apoptotic cell death during EAE were increased by MT-I+II deficiency. We now show for the first time that demyelination and axonal damage are significantly increased in MT-I+II deficient mice during EAE. Furthermore, oligodendroglial regeneration, growth cone formation, and tissue repair including expression of trophic factors were significantly reduced in MT-I+II-deficient mice during EAE. Accordingly, MT-I+II have protective and regenerative roles in the brain. Received 31 October 2002; received after revision 23 November 2002; accepted 26 November 2002 RID="*" ID="*"Corresponding author. M. Penkowa and C. Espejo contributed equally to this paper.     

小鼠金属硫蛋白基因-I 在CHO细胞中稳定表达及其在医疗方面的应用

将小鼠的MT-I基因插入pSV₂-dhfr质粒的EcoRI位点,构建了具有SV₄₀和MT双启动子并正向插入MT-I基因的表达载体。用改良的磷酸钙/DNA沉淀法将表达载体导入CHO-dhfr-细胞内,用不含次黄嘌呤(H)和胸腺嘧啶(T) 的选择培养基筛选出稳定表达MT阳性克隆D-22,经检测10⁶细胞的 MT 表达量约为1.8 μg,D-22细胞对Cd²⁺浓度抗性较之CHO-dhfr-细胞高14倍。为了研究金属硫蛋白对正常细胞所具有的保护作用,将不同浓度的顺铂分别处理无MT表达的CHO-dhfr-细胞和有MT表达的D-22细胞。实验发现顺铂对D-22细胞和CHO-dhfr^-细胞 50%生长抑制浓度(IC50)分别是0.145μmol/L 和0.04μmol/L。上述研究表明MT对正常细胞有一定的保护作用,因而通过诱导正常细胞提高MT的表达量,同时使用MT阻断剂阻断肿瘤组织的MT合成,这样可能会为治疗某些顽固肿瘤开辟一条新途径。     

金属硫蛋白(MT)的分离纯化与检测技术

综述了近年来在金属硫蛋白研究中应用的分离纯化和检测方法，对各种方法的适用性等特点作了评述。     

The enhanced induction of metallothionein by zinc,its half-life in the marine fishPleuronectes platessa,and the influence of stress factors on metallothionein levels

Summary Intraperitoneal injection of zinc raised levels of a hepatic metallothionein-like species. Assuming that this species was metallothionein (MT) then levels were raised from approximately 20 g/g to 300 g/g in 7 days, and levels thereafter remained high for the next 4 weeks. The half-lives of the protein in liver and kidney from starved fish, measured using in vivo incorporation of³⁵S cysteine at 11°C, were approximately 27 days and 32 days respectively. The following agents failed to stimulate synthesis of MT in plaice: stress (due to catching), endotoxin, dexamethasone, cortisol and turpentine.     

Restraint stress induced changes in rat liver and serum metallothionein and in Zn metabolism

Summary 24 h of a psychogenic stress (restraint) caused a strong increase of liver metallothionein (MT) levels. 3 h of stress were sufficient to induce an increase in liver MT, measured 21 h later, but the increase was much lower than in continuously restrained rats. Stress induction of liver MT was not due to food deprivation, since rats deprived for 24 h showed lower MT levels than stressed ones. Zn on MT presented the same qualitative but not quantitative pattern of response as MT protein. Liver cytosolic Zn was increased by restraint in spite of their being no decrease in serum Zn. Any treatment altered serum MT. Liver and serum MT were not correlated. The present results demonstrate that basically psychogenic stresses increased liver but not serum MT levels. No positive evidence for a relationship between corticosterone secretion and MT induction was found.     

金属硫蛋白的生理作用及在医学上的应用

本文简述了金属硫蛋白参与生物体内重金属离子代谢,消除重金属毒性,清除自由基和调节动物某些生理过程的作用以及在癌症治疗,抗肿瘤,抗辐射等方面的应用。     

^99Tc^m通过金属硫蛋白标记抗肿瘤单克隆抗体的研究

本文系统研究了^99Tc^m通过双功能联接剂金属硫蛋白（MT）标记抗肿瘤单克隆体的化学问题，并发展了新的实验方法和技术。用交联剂戊二醛制备MT与抗体（Ab)偶联物（MT－Ab)，偶联率为58％。用还原标记法制备^99Tc-MT-Ab，放射性利用率高达90％以上，而非特异标记低于10％。体外试验表明，在强络合剂竞争时，标记物有较高的稳定性。抗结肠癌单抗CL－3标记物的免疫反应活性保留87％。     

Recombinant synthesis of mouse Zn3-β and Zn4-α metallothionein 1 domains and characterization of their cadmium(II) binding capacity

Genetic engineering, coupled with spectro scopic analyses, has enabled the metal binding proper ties of the α and β subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn₄-αMT and Zn₃-βMT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to α fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the na tive protein. Titration of Zn₃-βMT with CdCl₂ results in the formation of Cd₃-βMT. The addition of excess Cd(II) leads to Cd₄-βMT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd₉-βMT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd₃-βMT species is stable in the presence of this strong metal-chelating agent. Received 20 May 1997; received after revision 7 July 1997; accepted 9 July 1997     

利用菲律宾花蛤的金属硫蛋白作为镉污染的检测指标

以菲律宾花蛤Ruditapes philippin Adams et Reave为实验动物,分别提取其外套膜和内脏团等组织的金属硫蛋白,经Sephadex G-75葡聚糖柱层析纯化后,用DV-600紫外可见分光光度计测定每个样品金属硫蛋白的吸收值,以研究菲律宾花蛤体内的金属硫蛋白浓度与镉污染之间的关系.结果表明:诱导实验能使检测菲律宾花蛤的内脏团和外套膜的金属硫蛋白含量均明显增加;金属硫蛋白的吸收值与菲律宾花蛤重量、壳长、壳宽均有较大的相关性.从这些关系中,认为利用金属硫蛋白作为菲律宾花蛤镉污染的检测指标是可行的.     

单扫极谱法在金属硫蛋白研究中的应用

本文将络合物吸附波测锌和镉用于金属硫蛋白(MT)的研究。在含α、α′-联吡啶介质中对经硝酸酸化后的MT样品中的锌含量进行测定,并将此法用于具有药用价值的Zn-MT的分离纯化及鉴定。在pH2的HCl-KI介质中,将单扫极谱法测镉用于镉-血红蛋白饱和法测定正常或金属诱导动物组织中的MT含量,检测量可达5μgMT/g组织,回收率为90～110％。