Effect of metal-based anticancer drugs on wild type and metallothionein null cell lines

Metallothioneins (MT) are ubiquitous low-molecular-weight metal-binding intracellular proteins. We used wild type mouse embryo fibroblasts, GKA1, and its MT-null variant, named GKA2, in order to correlate the presence of MT to the response to a number of different antitumor drugs with different mechanisms of action. We studied sensitivity of GKA1 and GKA2 cells to metal-based compounds having alkylating property, or able to generate reactive oxygen species (ROS); as well as to drugs acting with different mechanisms. The absence of MT in GKA2 cells was correlated to higher sensitivity to the metal-based drugs compared to that of GKA1. No marked differences in sensitivity of two cell lines against gemcitabine, taxol, and vinblastine were observed. No significant change in sensitivity of either GKA1 or GKA2 cells to these non-alkylating drugs was seen after heavy metal pretreatments. In GKA1 cells, MT biosynthesis was induced by copper and cadmium but not by zinc treatment under the conditions of these experiments. Induction of MT was directly proportional to decrease in sensitivity of GKA1 cells to the compounds used in this experiment. In contrast to GKA1 cells, the MT-null cells (GKA2) expressed no detectable metallothionein either constitutively or after treatment with zinc, copper, or cadmium. Nonetheless, heavy metal pretreatment of GKA2 cells did not cause any change in their sensitivity.     

Cd (II) stress response during the growth of Aspergillus niger B 77

Aims:  To investigate the relationship between growth, heavy metal ions uptake and participation of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in the protection of Apergillus niger B 77 against cadmium stress.
Methods and Results:  The stress response of the model fungal strain, under conditions of a wide range of Cd (II) ion concentrations, was investigated by determining the biomass formation, protein biosynthesis, SOD and CAT activities and heavy metal uptake in growing cells. Exposure to heavy metal ions induced an increase in protein content, heavy metal uptake and SOD activity, and a heavy decrease in CAT activity.
Conclusion:  The results obtained indicated that the tolerance of A. niger to Cd (II) was correlated with the heavy metal uptake, reactive oxygen species generation in the cells and the efficiency of antioxidative defence system.
Significance and Impact of the Study:  Evidence is provided for the possibility that oxidative stress plays a major role in the effect of Cd (II) ions on A. niger . These data could offer useful information when creating new strategies and methodological improvements for bioremediation with the participation of fungi.     

Plant metallothioneins – metal chelators with ROS scavenging activity?

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins present in plants, animals, fungi and cyanobacteria. In plants, MTs are suggested to be involved in metal tolerance or homeostasis, as they are able to bind metal ions through the thiol groups of their cysteine residues. Recent reports show that MTs are also involved in the scavenging of reactive oxygen species (ROS). The interplay between these roles is not entirely clear. Plants have many MT isoforms with overlapping expression patterns, and no specific role for any of them has been assigned. This review is focused on recent findings on plant MTs.     

基于活性氧自由基水平评价蔬菜重金属污染

蔬菜细胞中活性氧自由基水平处于一定范围内,而蔬菜一旦受重金属污染,活性氧自由基水平将发生改变.本文采用2＇,7＇-二氯荧光黄双乙酸酯标记蔬菜叶片细胞内活性氧自由基,激光扫描共聚焦显微镜技术分析蔬菜受重金属污染后活性氧自由基荧光强度的变化.结果表明,不同浓度镉、铅、汞离子（0、25、50、100 mg/L）作用芹菜、菠菜和油菜后,蔬菜叶片中活性氧自由基荧光强度均呈上升趋势,基于活性氧自由基水平的升高可判断蔬菜是否受重金属污染.本研究建立了基于活性氧自由基水平的蔬菜重金属污染评价方法,进一步为蔬菜的监管提供方法学和技术理论基础.     

Binding of transition metal ions [cobalt, copper, nickel and zinc] with furanyl-, thiophenyl-, pyrrolyl-, salicylyl- and pyridyl-derived cephalexins as potent antibacterial agents

A method is described for the preparation of novel cephalexin-derived furanyl-, thiophenyl-, pyrrolyl-, salicylyl- and pyridyl-containing compounds showing potent antibacterial activity. The binding of these newly synthesized antibacterial agents with metal ions such as cobalt(II), copper(II), nickel(II) and zinc(II) has been studied and their inhibitory properties against various bacterial species such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae are also reported. These results suggest that metal ions to possess an important role in the designing of metal-based antibacterials and that such complexes are more effective against infectious diseases compared to the uncomplexed drugs.     

Influence of zinc ions on protein secretion in a heavy metal tolerant strain of the ericoid mycorrhizal fungas Oidiodendron maius

A heavy metal tolerant strain of the ericoid mycorrhizal species Oidiodendron maius, isolated from soil heavily contaminated with zinc, was previously shown to tolerate high concentrations of zinc and cadmium ions in the growth medium. We have investigated some of the specific molecular responses of this fungal strain to the presence of increasing concentrations of zinc ions in the growth medium. In particular, we show that zinc ions induce a general change in the array of secreted proteins, with a shift towards the production of more basic, low molecular weight polypeptides. Some of these proteins were microsequenced and identified through homology search in databases. Among them are hydrolytic enzymes (nuclease, proteinase, lysozyme) and two superoxide dismutase isoforms. The latter are antioxidant enzymes known to play a role in heavy metal response in plants, animals and microorganisms.     

Binding of Transition Metal Ions [Cobalt,Copper, Nickel and Zinc] with Furanyl-, Thiophenyl-, Pyrrolyl-, Salicylyl-and Pyridyl-Derived Cephalexins as Potent Antibacterial Agents

A method is described for the preparation of novel cephalexin-derived furanyl-, thiophenyl-, pyrrolyl-, salicylyl- and pyridyl-containing compounds showing potent antibacterial activity. The binding of these newly synthesized antibacterial agents with metal ions such as cobalt(II), copper(II), nickel(II) and zinc(II) has been studied and their inhibitory properties against various bacterial species such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae are also reported. These results suggest that metal ions to possess an important role in the designing of metal-based antibacterials and that such complexes are more effective against infectious diseases compared to the uncomplexed drugs.     

Does vitamin C act as a pro-oxidant under physiological conditions?

Vitamin C readily scavenges reactive oxygen and nitrogen species and may thereby prevent oxidative damage to important biological macromolecules such as DNA, lipids, and proteins. Vitamin C also reduces redox active transition metal ions in the active sites of specific biosynthetic enzymes. The interaction of vitamin C with 'free', catalytically active metal ions could contribute to oxidative damage through the production of hydroxyl and alkoxyl radicals; whether these mechanisms occur in vivo, however, is uncertain. To examine this issue, we reviewed studies that investigated the role of vitamin C, both in the presence and absence of metal ions, in oxidative DNA, lipid, and protein damage. We found compelling evidence for antioxidant protection of lipids by vitamin C in biological fluids, animals, and humans, both with and without iron cosupplementation. Although the data on protein oxidation in humans are sparse and inconclusive, the available data in animals consistently show an antioxidant role of vitamin C. The data on vitamin C and DNA oxidation in vivo are inconsistent and conflicting, but some of the discrepancies can be explained by flaws in experimental design and methodology. These and other important issues discussed here need to be addressed in future studies of the role of vitamin C in oxidative damage.     

植物金属硫蛋白及其重金属解毒机制研究进展

金属硫蛋白是一类分子量较小、富含Cys的金属结合蛋白,广泛分布于生物界。近年来从植物中克隆到许多编码金属硫蛋白的基因,并在研究基因表达模式、组织表达特异性以及基因结构,如启动子、内含子在染色体上的定位等方面取得了一定进展,但对其功能的研究还处于起步阶段。很多实验表明,植物金属硫蛋白可以通过其大量的Cys残基螯合重金属并清除活性氧,使植物避免氧化损伤。文章介绍了植物金属硫蛋白的分类、特征、基因结构及其在植物重金属解毒中的作用。      

Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction

Divalent metal ions play a crucial role in catalysis by many RNA and protein enzymes that carry out phosphoryl transfer reactions, and defining their interactions with substrates is critical for understanding the mechanism of biological phosphoryl transfer. Although a vast amount of structural work has identified metal ions bound at the active site of many phosphoryl transfer enzymes, the number of functional metal ions and the full complement of their catalytic interactions remain to be defined for any RNA or protein enzyme. Previously, thiophilic metal ion rescue and quantitative functional analyses identified the interactions of three active site metal ions with the 3'- and 2'-substrate atoms of the Tetrahymena group I ribozyme. We have now extended these approaches to probe the metal ion interactions with the nonbridging pro-S(P) oxygen of the reactive phosphoryl group. The results of this study combined with previous mechanistic work provide evidence for a novel assembly of catalytic interactions involving three active site metal ions. One metal ion coordinates the 3'-departing oxygen of the oligonucleotide substrate and the pro-S(P) oxygen of the reactive phosphoryl group; another metal ion coordinates the attacking 3'-oxygen of the guanosine nucleophile; a third metal ion bridges the 2'-hydroxyl of guanosine and the pro-S(P) oxygen of the reactive phosphoryl group. These results for the first time define a complete set of catalytic metal ion/substrate interactions for an RNA or protein enzyme catalyzing phosphoryl transfer.     

高等植物重金属耐性与超积累特性及其分子机理研究

由于重金属污染日益严重, 重金属在土壤_植物系统中的行为引起了人们的高度重视。高等植物对重金 属的耐性与积累性, 已经成为污染生态学研究的热点。近年来, 由于分子生态学等学科的发展, 有关植物对重金属的解毒和耐性机理、重金属离子富集机制的研究取得了较大进展。高等植物对重金属的耐性和积累在种间和基因型之间存在很大差异。根系是重金 属等土壤污染物进入植物的门户。根系分泌物改变重金属的生物有效性和毒性, 并在植物吸收重金属的过程中发挥重要作用。土壤中的大部分重金属离子都是通过金属转运蛋白进入根细胞, 并在植物体内进一步转运至液泡贮存。在重金属胁迫条件下植物螯合肽 (PC) 的合成是植物对胁迫的一种适应性反应。耐性基因型合成较多的PC, 谷胱甘肽 (GSH) 是合成PC的前体, 重金属与PC螯合并转移至液泡中贮存, 从而达到解毒效果。金属硫蛋白 (MTs) 与PC一样, 可以与重金属离子螯合, 从而降低重金属离子的毒性。该文从分子水平上论述了根系分泌物、金属转运蛋白、MTs、PC、GSH在重金属耐性及超积累性中的作用, 评述了近 10年来这方面的研究进展, 并在此基础上提出存在的问题和今后研究的重点。     

Oxidative stress and signal transduction in Saccharomyces cerevisiae: insights into ageing, apoptosis and diseases.

In yeast, as in higher eukaryotes, reactive oxygen species are produced as normal by-products of cellular metabolism. Under physiological conditions, the cell defence mechanisms are able to avoid molecular damages. This balance is disturbed when yeast cells are exposed to diverse environmental stress conditions, such as the presence of oxidants, heat shock, ethanol and metal ions. The increased production of reactive oxygen species is sensed by the cell, leading to the induction of defence mechanisms - the oxidative stress response. The present review discusses the mechanisms by which reactive oxygen species are sensed and the signalling pathways that are coupled with changes in genomic expression programs. Yeast has been used as an eukaryotic cell system to characterise the molecular mechanisms underlying the oxidative stress response. Furthermore, yeast has been utilised to elucidate the role of oxidative stress in ageing, apoptosis, and diseases, such as familial amyotrophic lateral sclerosis and Friedreich's ataxia.     

Interaction of phospholipid-metal complexes with water-soluble wheat protein

Insoluble lipid-protein complexes are formed in the presence of Ni(II), Ca(II), or Mg(II) by specific components of the water-soluble proteins of wheat flour and either triphosphoinositide or phosphatidyl serine. The pattern of protein species bound by the lipid-metal complex is dependent upon the metal and the phospholipid used. A group of proteins, containing carbohydrate, may be solubilized and recovered by washing the precipitate with acidic chloroform-methanol-water. Analyses of reactive and nonreactive protein species have shown no differences which clearly account for their behavior. Methylation of protein increases binding to lipid; acetylation decreases the interaction. Weak interaction has been observed between certain components of flour proteins and phospholipid in the absence of metal ions, but the components differ from those bound in the presence of metal ions. It is suggested that properly oriented groups of the protein molecules are chelating onto available coordination positions of metal ions already bound to phospholipid.     

An iron hydroxide moiety in the 1.35 Å resolution structure of hydrogen peroxide derived myoglobin compound II at pH 5.2

The biological conversions of O(2) and peroxides to water as well as certain incorporations of oxygen atoms into small organic molecules can be catalyzed by metal ions in different clusters or cofactors. The catalytic cycle of these reactions passes through similar metal-based complexes in which one oxygen- or peroxide-derived oxygen atom is coordinated to an oxidized form of the catalytic metal center. In haem-based peroxidases or oxygenases the ferryl (Fe(IV)O) form is important in compound I and compound II, which are two and one oxidation equivalents higher than the ferric (Fe(III)) form, respectively. In this study we report the 1.35 A structure of a compound II model protein, obtained by reacting hydrogen peroxide with ferric myoglobin at pH 5.2. The molecular geometry is virtually unchanged compared to the ferric form, indicating that these reactive intermediates do not undergo large structural changes. It is further suggested that at low pH the dominating compound II resonance form is a hydroxyl radical ferric iron rather than an oxo-ferryl form, based on the short hydrogen bonding to the distal histidine (2.70 A) and the Fe...O distance. The 1.92 A Fe...O distance is in agreement with an EXAFS study of compound II in horseradish peroxidase.     

Prion metal interaction: Is prion pathogenesis a cause or a consequence of metal imbalance?

Functional role of cellular prion protein (PrPc) has been hypothesized to be in metal homeostasis and providing cells with a superoxide dismutase (SOD)-like activity to escape damage by reactive oxygen species (ROS). PrPc interacts with a range of divalent metal ions and undergoes Cu²⁺ as well as Zn²⁺-associated endocytosis, thereby maintaining homeostasis of these and other metal ions. Conformational change to a β-sheet rich, protease resistant entity, reminiscent of the disease-associated scrapie form called PrPsc, has been found to be induced by interaction of PrPc with metal ions like Cu²⁺, Zn²⁺, Mn²⁺ and Fe²⁺. This review compiles data from various experimental studies of the interaction of metals with PrPc. The effect of metal ions on the expression and conformation of the prion protein is described in detail with emphasis on their possible physiological and pathogenic role. Further, a hypothesis is presented where attainment of altered conformation by metal-bound PrPc has been viewed as a deleterious consequence of efforts made by cells to maintain metal homeostasis. Thus, PrPc presumably sacrifices itself by converting into PrPsc form in an attempt to protect cells from the toxicity of metal imbalance. Finally, possible reasons for contradictions reported in the literature on the subject are explored and experimental approaches to resolve the same are suggested.     

植物对重金属耐性的分子生态机理

植物适应重金属元素胁迫的机制包括阻止和控制重金属的吸收、体内螯合解毒、体内区室化分隔以及代谢平衡等。近年来,随着分子生物学技术在生态学研究中的深入应用,控制这些过程的分子生态机理逐渐被揭示出来。菌根、根系分泌物以及细胞膜是控制重金属进入植物根系细胞的主要生理单元。外生菌根能显著提高寄主植物的重金属耐性,根系分泌物通过改变根际pH、改变金属物质的氧化还原状态和形成络合物等机理减少植物对重金属的吸收。目前,控制菌根和根系分泌物重金属抗性的分子生态机理还不清楚。但细胞膜跨膜转运器已得到深入研究,相关金属离子转运器被鉴定和分离,一些控制基因如铁锌控制运转相关蛋白(ZIP)类、自然抵抗相关巨噬细胞蛋白(Nramp)类、P_1B-type ATPase类基因已被发现和克隆。金属硫蛋白(MTs)、植物螯合素(PCs)、有机酸及氨基酸等是植物体内主要的螯合物质,它们通过螯合作用固定金属离子,降低其生物毒性或改变其移动性。与MTs合成相关的MT-like基因已经被克隆,PCs合成必需的植物螯合素合酶(PCS), 即γ-Glu-Cys二肽转肽酶(γ-ECS) 的编码基因已经被克隆,控制麦根酸合成的氨基酸尼克烟酰胺(NA)在重金属耐性中的作用和分子机理也被揭示出来。ATP 结合转运器(ABC)和阳离子扩散促进器(CDF) 是植物体内两种主要膜转运器,通过它们和其它跨膜方式,重金属被分隔贮藏于液泡内。控制这些蛋白转运器合成的基因也已经被克隆,在植物中的表达证实其与重金属的体内运输和平衡有关。热休克蛋白(HSP)等蛋白类物质的产生是一种重要的体内平衡机制,其分子机理有待进一步研究。重金属耐性植物在这些环节产生了相关响应基因或功能蛋白质,分子克隆和转基因技术又使它们在污染治理上得到了初步的应用。