WTC rat has unique characteristics such as resistant to streptozotocin

Because we found that WTC rats might be resistant to streptozotocin (STZ), we have elucidated the mechanisms of resistant to the diabetogenic effects of STZ in the WTC rats. Dose response to STZ was evaluated with glucose levels. No significant changes in glucose level to STZ administration were observed in WTC rats. Insulin secretion by suppling glucose was preserved in WTC rats even after STZ administration. Although there was no significant difference in gene expression of both GLUT2 and Kir6.2, which were involved in STZ resistance, between WTC rats and Wistar rats, the expression of metallothionein 2a in pancreas and liver to STZ administration of WTC rats was significantly higher than that of Wistar rats. Moreover, alloxan did not induce diabetes in WTC rats as same as STZ. These results suggest that WTC rats might have powerful antioxidant property to protect β cells in pancreas. Because the STZ-resistant property is very close characteristics to human beings, WTC rats will become a useful animal model in diabetic researches.     

Metallothionein responses in the Asiatic clam (Corbicula fluminea) after exposure to trivalent arsenic

The main objective of this work was to evaluate arsenic effects on metallothionein (MT) induction by exposing a freshwater Asiatic clam (Corbicula fluminea) to different concentrations of this metalloid. The presence of MT-like proteins was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and compared with a standard rabbit MT. In addition, the polarographic response showed good correspondence between standard MT and MT-like curves from C. fluminea, allowing MT quantification. The results show that clams exposed to different concentrations of arsenic are able to induce significant levels of MTs. Although variability was found in MT induction, significant differences in MT levels were found after 28 days of exposure in all treatments in comparison with the controls, suggesting that exposure to arsenic induced MT-like proteins in C. fluminea.     

Metallothionein concentration in the mussel Mytilus galloprovincialis as a biomarker of response to metal contamination: validation in the field

Mussels were translocated from a shell-fish breeding area (Sète, on the French Mediterranean coast) to sites exposed to trace element inputs in April 2000. They were recovered 3 months later. Whole soft tissues from all of the sites (n = 97) were analysed for arsenic, cadmium, chromium, copper, mercury, nickel, lead and zinc. Metallothioneins (MTs) were also measured in the digestive gland and in the remaining tissues (allowing calculation of whole soft tissue concentrations) at 22 of the 97 sites. MT concentrations in the digestive gland and the whole soft tissues were strongly correlated. The condition index varied with food availability at different sites. This did not influenced MT concentrations in the whole soft tissues, whereas the condition index was negatively correlated to trace element concentrations. A model is proposed to minimize this influence of condition. Metal concentrations adjusted using this model showed significant correlations with MT levels for those metals (cadmium, copper, nickel and zinc) that are known to bind to this protein, with the exception of mercury. Even in moderately contaminated sites, measurement of the MT level in the soft tissues of mussels was generally able to discriminate between different levels of contamination, allowing the use of a simplified procedure compared with dissection of the digestive gland. It is recommended to avoid translocation and sampling during the reproductive period, which is well documented for commercial species such as Mytilus sp.     

Application of two SH-based methods for metallothionein determination in mussels and intercalibration of the spectrophotometric method: laboratory and field studies in the Mediterranean Sea

Metallothionein (MT) induction is widely used as a biomarker of exposure to metals in mussels. The aims of the present work were first to compare the suitability of spectrophotometry and differential pulse polarography (DPP) for MT detection in mussels exposed to 200 ppb cadmium for 9 days in a laboratory experiment and in mussels sampled in different seasons from expected pollution gradients along the Mediterranean Sea; second, to intercalibrate the widely used spectrophotometric method using mussels from Saronikos Gulf. In the intercalibration of the spectrophotometric method, similar results (p>0.05) were obtained by two different research teams indicating a good reproducibility of the technique. However, polarographic and spectrophotometric methods gave significantly (p<0.05) different results in laboratory and field studies. In the laboratory experiment, MT values detected with DPP were nine times higher than with spectrophotometry. The results obtained by the two methods were significantly correlated. Both methods could discriminate between control and exposed mussels. In field studies, MT values obtained by DPP were 34–38-fold higher than with spectrophotometry, and MT concentrations measured by both methods were not correlated. This discrepancy could be due to several factors, including the low levels of bioavailable metals in the studied areas and the possibility that the different methods can measure MT isoforms differentially. Further work is needed to decipher the functions of MT isoforms in mussels. This information is relevant for the application of MT as a biomarker in biomonitoring programmes.     

斧文蛤金属硫蛋白基因的克隆与表达分析

金属硫蛋白（Metallothionein,MT）是一类富含半胱氨酸的小分子蛋白质,参与机体重金属解毒、维持金属元素代谢平衡以及清除自由基等生理功能。为了解斧文蛤金属硫蛋白（Ml-MT）的分子生物学特征及其在重金属Cd2+胁迫下的响应机制,本文采用RACE技术从斧文蛤（Meretrix lamarckii）总RNA反转录产物中获得了636 bp的Ml-MT cDNA基因序列。该序列包含65 bp的5非编码区（UTR）和340 bp的3非编码区（UTR）以及231 bp的开放阅读框（ORF）,可编码76个氨基酸;其中半胱氨酸占27%,不含芳香族氨基酸,含16个MT所特有的Cys-Xn-Cys结构,预测的分子量约为7.704 kD,理论等电点7.138。MT氨基酸序列比对分析表明:斧文蛤金属硫蛋白（Ml-MT）与丽文蛤（Meretrix lusoria）的相似性高达88%,与文蛤（Meretrix meretrix）的同源性为87%。实时荧光定量（qRT-PCR）检测MT在斧文蛤5种组织中均有表达,但存在组织特异性,其中内脏团表达量最高,其次依次为鳃丝、闭壳肌、外套膜、斧足。在Cd2+（0.13 mg/L）胁迫0、6h、12h、24h、48h、72h和96h下,斧文蛤内脏团MT呈现出不同程度的上调表达,具体表现为高-低-高-低的波浪式变化,除6h以外,其他时间点均与对照组存在极显著差异（P0.01）。本研究表明:MT基因在维护机体正常生理功能及斧文蛤抵御重金属Cd2+胁迫过程中发挥着重要的分子调控作用。     

Developmental variation in copper,zinc and metallothionein mRNA in brindled mutant and nutritionally copper deficient mice

The concentrations of copper, zinc and metallothionein-I (MT-I) mRNA were determined in the liver, kidney and brain of the brindled mutant mouse from birth until the time of death. Despite accumulation of copper in the kidney of the mutant, MT-I mRNA concentrations were normal. There was no difference between the MT-I mRNA in the brain of mutant and normal in the first 10 days of life, but after day 10 metallothionein mRNA levels were increased in the mutant. The concentration of copper was very low in the liver of the mutant, and on day 6 after birth the metallothionein mRNA was also reduced by about 50%. This reduction was not seen in copper-deficient 6-day-old pups, despite very low hepatic copper levels. This suggests that the lower hepatic MT-I mRNA in the day 6 brindled mouse was not simply due to the reduction in hepatic copper and also that hepatic copper is not regulating metallothionein gene expression the liver of neonatal mice. After day 12 hepatic MT-I mRNA levels were elevated in mutant and in copper deficient mice, both of which die at 14 to 16 days. These increases and the increase in brain MT-I mRNA in older mutant mice are likely to be caused by stress. Overall the results support the conclusions that the brindled mutation does not cause a constitutive activation of the metallothionein genes, and that the differences in metallothionein mRNA between mutant and normal are most probably secondary consequences of the mutation.     

力竭游泳诱导的大鼠睾丸金属结合蛋白的初步分离与鉴定

目的初步分离和鉴定力竭运动诱导的大鼠睾丸金属结合蛋白(testis metal-binding proteins,TMB-Ps)。方法 8只雄性SD大鼠一次性力竭游泳运动(8只对照)后6 h取睾丸和肝脏组织,用镉饱和法测定TMBPs和肝脏金属硫蛋白(metallothionein,MT)含量,用tricine-SDS-PAGE方法分离镉饱和法提取液的TMBPs组分和肝脏MT,用液相色谱-串联质谱法(LC-MS-MS)鉴定TMBPs分离条带。结果力竭运动组大鼠TMBPs水平(113.71±11.72)nmol Cd/g testis显著高于安静对照组(87.14±12.72)nmol Cd/g testis(P0.01),肝脏MT表达量(64.70±14.89)μg/g也显著高于安静对照组(7.32±3.31)μg/g(P0.001)。LC-MS-MS对tricine-SDS-PAGE分离的蛋白条带的鉴定结果为:TMBPs含有泛素、铜-锌-超氧化物歧化酶、酪蛋白样磷蛋白等蛋白质,另外还应包括MT。结论TMBPs由一组具有金属结合性、耐热性和诱导性的蛋白质所组成,主要有泛素、超氧化物歧化酶和酪蛋白样磷蛋白。镉饱和法并非测定金属硫蛋白的特异性方法。     

人金属硫蛋白在乳酸菌中的融合表达及其预防食源性镉污染的应用

目的:镉(Cd)是一种重要的环境污染物,其具有半衰期长、不能生物降解等特性使之易于在人体重要脏器中蓄积并对人身体健康造成不可逆的损伤。为此,减少镉的摄入,尤其是经胃肠道,是预防人体慢性镉中毒的有效方法。方法:构建以α-半乳糖苷酶作为筛选标记、表达金属硫蛋白融合蛋白(GST-SUMO-MT)的重组乳酸乳球菌MG1363/p M-GSMT。原子吸收光谱法分析重组乳酸菌对Cd~(2+)和Zn~+的结合能力。以灌胃方式,对雄性Sprague-Dawley大鼠每天口服不同剂量的重组乳酸菌(2×10~9~2×10~(11)CFU/d)及CdCl_2溶液[5mg/(kg·d)],连续处理56天。收集实验动物肝、肾、脑及睾丸,利用原子吸收光谱法检测这些脏器中镉离子的含量。生化分析检测血清中尿素及丙氨酸转氨酶的含量。石蜡切片及苏木精-伊红染色分析各组织的病理变化。结果:获得不含抗生素抗性的重组乳酸菌MG1363/p M-GSMT,其吸附Cd~(2+)、Zn~(2+)能力比对照组分别提高3.52倍和1.60倍。动物实验结果显示,镉能在肝、肾、脑及睾丸中蓄积,并对这些器官造成明显的病理损伤。原子吸收光谱分析、血清生化指标及病理切片结果都显示,重组乳酸菌能有效降低胃肠道中镉的摄入,进而减少Cd~(2+)在各脏器中的蓄积及对器官功能的损伤。结论:为预防人体食源性慢性镉中毒提供了一种有效的生物材料和使用方法。