Metallothionein as a tool in biomonitoring programmes

The biochemical features of metallothioneins and their functional role in the cell are described. On this basis, the potential role of MTs as a biomarker of exposure in aquatic organisms, such as fishes and molluscs, is evaluated in the light of recent knowledge about MT gene regulation and inducibility. It appears that in fish MTs should be considered as a kind of stress protein which is particularly responsive to heavy metals. In molluscs, in particular in mussels, MTs seem more specifically involved in responses to heavy metals and they should therefore be considered a biomarker of exposure to heavy metal pollution. Common techniques for MT evaluation are listed and a simple spectrophotometric method recently developed is also reported. Finally, the correct approach to the use of MTs as a biomarker of exposure in biomonitoring programmes for an assessment of the physiological status of aquatic organisms is discussed.     

花生金属硫蛋白基因AhMT3a的克隆及其表达

以鲁花14号花生为材料,从花生cDNA文库和基因组中筛选和克隆了花生的金属硫蛋白基因AhMT3a。该基因全长785 bp,有2个内含子,开放阅读框由201个碱基组成,编码66个氨基酸,其中包含13个半胱氨酸(Cys),预测其分子量为6.83kD,等电点为4.59。运用生物信息学手段对AhMT3a蛋白的信号肽、跨膜区、亚细胞定位和疏水性进行了预测。与拟南芥、棉花和草莓等植物type 3 MTs的序列比对结果表明,花生和其他不同植物的MT3在氨基酸序列上具有较高的同源性,从系统发育树中可以看出AhMT3a和蒿麦的金属硫蛋白亲缘关系较近。半定量RT-PCR和芯片杂交结果显示花生AhMT3a在花中表达量最高,在种子中表达量最低;在ABA、NaCl及PEG等不同处理下,表达量变化不大。     

金属硫蛋白与医学

金属硫蛋白是一类富含巯基与金属的内源性蛋白,化学结构极为特殊,该蛋白的为具有很强的诱导性,金属,糖皮质激素,某些化的和应激状态均可诱导其生成,在炎症等应激过程中ＩＬ－６可能为诱导者。在不降低疗效的情况下该蛋白可解除抗肿瘤药物ＣＤＤＰ的抗药性密切有关,金属硫蛋白与炎性肠病,妊娠中毒症,肝脏疾患等多种疾病密切相关。     

紫羊茅重金属抗性和敏感品种中类金属硫蛋白基因的鉴定及表达初探

重金属对生命机体的作用具有双重性。一方面,作为多数辅酶的辅助因子对细胞的正常代谢必不可少;另一方面,当重金属超过一定的浓度时对细胞有较大的毒性。在长期的进化过程中,生物可能形成一种调节细胞内重金属浓度的机制。这种机制在动物和真菌中被认为同金属硫蛋白(metallothionein,MT)的作用密切相关。植物中也存在类似的与重金属结合的低分子量蛋白(heavy metal binding pep-tide)。最近对拟南芥菜和水稻中类金属硫蛋白(MT-like)基因的研究证实其作用与动物MT相似。紫羊茅品种“Merlin”是一种从锌铅矿区的重金属污染地采集的单子叶草种,对镉和铜的抗性都较高,分别达到50mg/L和30mg/L,而5mg/L Cd~(2 )或2mg/L Cu~(2 )便可抑制敏感品种“S59”的正常生长。目前     

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。     

Expression, purification and preliminary characterization of mussel (Mytilus galloprovincialis) metallothionein MT20

Metallothioneins are rather ubiquitous metal-binding proteins induced by stressing or physiological stimuli. Two major metallothionein isoforms have been identified in mussel: MT10 and MT20. Nevertheless the high sequence homology, the two isoforms exhibit different expression and inducibility in vivo. We cloned and produced in Escherichia coli the MT20 isoform from Mytilus galloprovincilis. cDNA was subcloned into pGEX-6P.1 vector, in frame with a sequence encoding a glutathione-S-transferase (GST) tail. Recombinant protein was purified to electrophoretic homogeneity by affinity chromatography. After enzymatic cleavage of the GST tail the MT moiety was recovered with a final yield of about 5 mg of protein per litre of bacterial culture. The metal-binding ability of MT20 was assessed by absorption spectroscopy upon addition of cadmium equivalents and the metal release was checked as a function of the environment pH. Moreover the protein was analysed for the propensity to polymerization, typical of this class of protein, before and after exposure to reducing and alkylating agents.     

嗜热四膜虫金属硫蛋白基因MMT2和MTT4的表达规律比较与进化模式分析

多个金属硫蛋白基因异构型已在四膜虫中被鉴定,这些异构型可分为7a和7b两个亚家族。该文利用实时荧光定量PCR技术检测了嗜热四膜虫金属硫蛋白基因MTT2和MTT4在Hg、Cu、Cd、Zn、H2O2暴露下的表达水平,结果显示两者表达规律相似,均为:Cu暴露下上调最高(>200倍),Hg次之,Cd、Zn上调倍数不大,H2O2有下调趋势。此表达规律明显有别于7a亚家族,具有7b亚家族的表达特征。同种诱导物暴露下MTT4的上调表达幅度比MTT2高出数倍,结合生物信息学分析结果,推测可能与MTT2和MTT4上游调控元件(如AP-1、MRE等)的数量差异有关。基于MTT2和MTT4在结构和功能上的高度相似性,推测两者可能是经近期基因复制事件产生,并遵循基因剂量模型进化而来。     

Role of Mechanical Stress-induced Glutamate Signaling-associated Molecules in Cytodifferentiation of Periodontal Ligament Cells

In this study, we analyzed the effects of tensile mechanical stress on the gene expression profile of in vitro-maintained human periodontal ligament (PDL) cells. A DNA chip analysis identified 17 up-regulated genes in human PDL cells under the mechanical stress, including HOMER1 (homer homolog 1) and GRIN3A (glutamate receptor ionotropic N-methyl-d-aspartate 3A), which are related to glutamate signaling. RT-PCR and real-time PCR analyses revealed that human PDL cells constitutively expressed glutamate signaling-associated genes and that mechanical stress increased the expression of these mRNAs, leading to release of glutamate from human PDL cells and intracellular glutamate signal transduction. Interestingly, exogenous glutamate increased the mRNAs of cytodifferentiation and mineralization-related genes as well as the ALP (alkaline phosphatase) activities during the cytodifferentiation of the PDL cells. On the other hand, the glutamate signaling inhibitors riluzole and (+)-MK801 maleate suppressed the alkaline phosphatase activities and mineralized nodule formation during the cytodifferentiation and mineralization. Riluzole inhibited the mechanical stress-induced glutamate signaling-associated gene expressions in human PDL cells. Moreover, in situ hybridization analyses showed up-regulation of glutamate signaling-associated gene expressions at tension sites in the PDL under orthodontic tooth movement in a mouse model. The present data demonstrate that the glutamate signaling induced by mechanical stress positively regulates the cytodifferentiation and mineralization of PDL cells.     

Variations in plant metallothioneins: the heavy metal hyperaccumulator <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis> as a study case

Plant metallothioneins (MTs) are extremely diverse and are thought to be involved in metal homeostasis or detoxification. Thlaspi caerulescens is a model Zn/Cd hyperaccumulator and thus constitutes an ideal system to study the variability of these MTs. Two T. caerulescens cDNAs (accession: 665511; accession: 665515), that are highly homologous to type 1 and type 2 Arabidopsis thaliana MTs, have been isolated using a functional screen for plant cDNAs that confer Cd tolerance to yeast. However, TcMT1 has a much shorter N-terminal domain than that of A. thaliana and so lacks Cys motifs conserved through all the plant MTs classified as type 1. A systematic search in plant databases allowed the detection of MT-related sequences. Sixty-four percent fulfil the criteria for MT classification described in Cobbett and Goldsbrough (2002) and further extend our knowledge about other conserved residues that might play an important role in plant MT structure. In addition, 34% of the total MT-related sequences cannot be classified strictly as they display modifications in the conserved residues according to the current plant MTs’ classification. The significance of this variability in plant MT sequences is discussed. Functional complementation in yeast was used to assess whether these variations may alter the MTs’ function in T. caerulescens. Regulation of the expression of MTs in T. caerulescens was also investigated. TcMT1 and TcMT2 display higher expression in T. caerulescens than in A. thaliana. Moreover, their differential expression patterns in organs and in response to metal exposure, suggest that the two types of MTs may have diverse roles and functions in T. caerulescens.     

Histochemical demonstration of heavy metal tolerance in ectomycorrhizal fungi

Summary By the use of a specific histochemical staining method evidence was obtained that tolerance to heavy metals in ectomycorrizal fungi is based on the presence of metallothionein-like proteins. The implication that tolerance in these fungi should be induced by sublethal concentrations of heavy metals has been confirmed by us. Induction of metallothionein in ectomycorrizal fungi could possibly be helpful in protection of their host plants in areas polluted by heavy metals. In comparison with biochemical methods the histochemical method is able to locate the metal tolerance and has the added advantage that it may also be applied to mycorrhizas (root and fungus).     

斑节对虾金属硫蛋白基因cDNA克隆与表达分析

采用RACE-PCR方法从斑节对虾(Penaeus monodon)总RNA反转录产物中获得了502 bp的金属硫蛋白(Pm-MT)基因cDNA序列。该序列包含69 bp 的5非编码区(UTR)和256 bp 的3非编码区(UTR)以及177 bp的开放阅读框(ORF), 可编码58个氨基酸。在其编码的氨基酸序列中半胱氨酸含量丰富, 不含芳香族氨基酸, 存在有无脊椎动物金属硫蛋白的特征序列CKCXXXCXCX, 预测的分子量约为6.05 kD, 理论等电点7.75。序列比对分析表明Pm-MT与北美淡水蟹(Pacifastacus leniusculus)的相似性高达91.4%, 与美国龙虾(Homarus americanus)的同源性最高为84.5%。实时定量PCR显示在所检测组织中, Pm-MT的mRNA在卵巢的表达量最高, 其次为胃、肠、心脏、脑和肝胰腺; Pm-MT的mRNA在斑节对虾6个不同发育期的卵巢中的表达量都很高, 其中在Ⅱ期卵巢中的表达量最高。实验所得结果以期为Pm-MT进一步在卵巢发育的功能研究提供基础材料。     

Variation in metallothionein gene expression is associated with adaptation to copper in the earthworm Dendrobaena octaedra

Evolution of resistance to heavy metals has been reported for several populations of soil living organisms occurring at metal contaminated sites. Such genetically based and heritable resistance contribute to the persistence of populations in contaminated areas. Here we report on molecular responses to experimental copper in populations of the earthworm, Dendrobaena octaedra, originating from copper contaminated soil near Gusum (Sweden) where heavy metal pollution has been present for several decades. We studied gene expression of six genes potentially involved in resistance to copper toxicity using F2-generations of D. octaedra populations, originating from reference sites and contaminated (High, Medium and Low) sites around Gusum. The main result was different expression patterns of genes encoding for two different isoforms (mt1 and mt2) of metallothionein proteins during experimental exposure to copper contaminated soil. Expression of mt1 showed a fast and significant upregulation in the High population and a slower, albeit significant, upregulation in Medium and Low populations. However, in the three reference populations no upregulation were seen. In comparison, a fast upregulation was also seen for the High population in the isoform mt2, whereas, gene expression of all other populations, including reference populations, showed slower upregulation in response to experimental copper. The results indicate that copper resistance in D. octaedra from contaminated areas is related to an increased expression of metallothioneins.     

Enhancement of MT synthesis by leptin in fasted mice

It is known that metallothionein (MT) synthesis occurs in the liver in various stressful situations such as immobilization and fasting. However, the mechanism of MT synthesis in stressful situations is not fully understood. In this study, we examined the involvement of leptin, the obese gene product, in MT synthesis induced by fasting stress. Despite an increase in hepatic MT levels induced by 24-hr fasting in wild-type mice, both wild-type and MT-null mice showed decreases in plasma leptin levels after 24 hr of fasting. Hepatic MT levels increased to levels comparable with that in control mice in ICR, C3H, 129Sv and Balb/c mice fasted for 24 hr, and plasma leptin levels decreased significantly. Repetition of fasting and feeding in turn every 24 hr caused a gradual decrease in hepatic MT levels after the fasting period. In contrast, the reduced plasma leptin levels increased after the fasting period with repetition of fasting-feeding cycles. The findings indicate that there is an adaptation to starvation. On the other hand, subcutaneous leptin infusion in fasted mice via an osmotic pump resulted in increases in hepatic MT levels compared to the levels in vehicle-treated mice after 24 hr of fasting. Only leptin infusion had no effect on hepatic MT levels. These results suggest that MT synthesis in fasting stress is not correlated with decrease in plasma leptin levels but that leptin itself is a potent inducer of MT in a fasting situation.     

Metallothionein and stress combine to affect multiple organ systems

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins that have a wide range of functions in cellular homeostasis and immunity. MTs can be induced by a variety of conditions including metals, glucocorticoids, endotoxin, acute phase cytokines, stress, and irradiation. In addition to their important immunomodulatory functions, MTs can protect essential cellular compartments from toxicants, serve as a reservoir of essential heavy metals, and regulate cellular redox potential. Many of the roles of MTs in the neuroinflammation, intestinal inflammation, and stress response have been investigated and were the subject of a session at the 6th International Congress on Stress Proteins in Biology and Medicine in Sheffield, UK. Like the rest of the cell stress response, there are therapeutic opportunities that arise from an understanding of MTs, and these proteins also provide potential insights into the world of the heat shock protein.