Zinc and N-acetylcysteine modify mercury distribution and promote increase in hepatic metallothionein levels

This study investigated the ability of zinc (Zn) and N-acetylcysteine (NAC) in preventing the biochemical alterations caused by mercury (Hg) and the retention of this metal in different organs. Adult female rats received ZnCl₂ (27 mg/kg) and/or NAC (5 mg/kg) or saline (0.9%) subcutaneously and after 24 h they received HgCl₂ (5 mg/kg) or saline (0.9%). Twenty-four hours after, they were sacrificed and analyses were performed. Hg inhibited hepatic, renal, and blood δ-aminolevulinic acid dehydratase (δ-ALA-D) activity, decreased renal total thiol levels, as well as increased serum creatinine and urea levels and aspartate aminotransferase activity. HgCl₂-exposed groups presented an important retention of Hg in all the tissues analyzed. All pre-treatments demonstrated tendency in preventing hepatic δ-ALA-D inhibition, whereas only ZnCl₂ showed this effect on blood enzyme. Moreover, the combination of these compounds completely prevented liver and blood Hg retention. The exposure to Zn and Hg increased hepatic metallothionein levels. These results show that Zn and NAC presented promising effects against the toxicity caused by HgCl₂.     

苹果金属硫蛋白基因MdFjMT2克隆及生物信息学分析

以红富士苹果（Malus domestica CV Red Fuji）浓红型芽变和其条红母株为试材,构建抑制性差减杂交（Suppression Subtractive Hybridization,SSH）文库,差异筛选SSH-cDNA文库,经过大量测序构建成红富士苹果ESTs序列本地数据库,对本地数据库进行Blastn检索,得到42条金属硫蛋白基因MdFjMT2 cDNA片段,通过序列拼接和?逆转录PCR（RT-PCR）方法,获得了红富士苹果金属硫蛋白基因MdFjMT2全长cDNA序列（Genbank登录号为HQ730757）,该基因全长684 bp,其中5′ 非翻译区97 bp,3′ 非翻译区347 bp,开放阅读框（ORF）为240 bp,编码79个氨基酸,推测蛋白分子量为7.7938 kDa,理论等电点为4.75。该基因具有金属硫蛋白基因的典型结构域特征,编码的氨基酸序列中含有14个半胱氨酸残基Cys（C）,Cys 残基的排列特征是以CC、CXC和CXXC集中分布在肽链的N端和C端。系统进化分析表明MdFjMT2与砂梨（Pyrus pyrifolia）、苹果（Malus domestica）和小金海棠（M. xiaojinensis）等植物金属硫蛋白保持了较近的亲缘关系,与扁桃（Prunus dulcis）、旱柳（Salix matsudana）、美味猕猴桃（Actinidia deliciosa）等植物的亲缘关系较远。生物信息学分析结果表明,MdFjMT2主要位于叶绿体中,没有信号肽,是非跨膜亲水性蛋白,其蛋白质二级结构的主要元件是无规则卷曲,没有功能结构域。这些结果为MdFjMT2的结构与功能挖掘提供一定的参考。本研究结果有助于研究该基因在苹果着色中的作用,阐明苹果着色的分子机制。     

Manipulations of metallothionein gene dose accelerate the response to Listeria monocytogenes

Metallothioneins (MTs) are cysteine-rich proteins that assist in cellular homeostasis and protect against oxidant injury. MTs can be induced by heavy metals and inflammatory mediators and function as free radical scavengers, reservoirs for essential heavy metals, and immunomodulators. In light of MTs’ roles in responses to stress, we evaluated the in vivo effects of MT gene dose on the course of Listeria monocytogenes (LM) infection. LM burden was measured in livers and spleens, and flow cytometric assays were used to analyze splenocyte surface sulfhydryls, oxidative burst and apoptosis. Our results suggest that deviations from the normal complement of MT genes alter the course of LM infection. Compared to the wild-type C57BL/6J (B6-WT) strain, a congenic partner that carries a larger number of Mt1 genes (B6-MTTGN) and a congenic strain in which both Mt1 and Mt2 are disrupted (B6-MTKO) both showed lower bacterial burdens three days post-inoculation. This difference was prominent in the first 48 h of infection, after which LM clearance occurred at comparable rates in all three strains. Lymphocytes from B6-MTKO mice exhibited increased cell death and increased levels of surface sulfhydryls compared to B6-WT and B6-MTTGN mice. Lymphocytes from B6-MTTGN mice had increased levels of intracellular oxidants compared to B6-WT and B6-MTKO mice. The oxidative burst by macrophages from infected B6-MTTGN and B6-MTKO mice was increased, suggesting one mechanism by which these strains might reduce the LM burden. These results indicate that MT gene dose dramatically influences host-defenses against LM infection.     

Population-related molecular responses on the effect of pesticides in Carassius auratus gibelio

The aim of this study was to evaluate population-related peculiarities of the adaptive responses of Carassius auratus gibelio. In order to do this, male specimens from polluted (B) and clean (Z) sites were exposed to commercial pesticides thiocarbamate Tatoo (9.1 μg·L(-1)and 91 μg·L(-1)) or tetrazine Apollo (2 μg·L(-1) and 10 μg·L(-1)) during fourteen days. The control fish from site B was distinguished by weakness of antioxidant defence (measured from superoxide dismutase and catalase activities, redox index of glutathione (GSH), superoxide anion (O(2)) and lipid peroxidation levels), imbalance of the concentrations of protein metallothionein (MT-SH) and MT-related metals (MT-Me) and neurotoxicity. Differences in glutathione-S-transferase activity in the liver and vitellogenin-like proteins in the serum were also showed between B and Z control groups. Common effects of pesticides were related to a decrease in GSH, an increase in O(2) production, ethoxyresorufin O-deethylase activity and hepatosomatic index. Apollo provoked particular elevation of MT-SH/MT-Me ratio. Population-related difference in the response was the activation of antioxidant defence in fish from site B and its inhibition in fish from site Z. The genotoxic effect of exposures was more expressed in fish from site B. Principal component analysis combine all exposed groups from site Z and control group from site B in one set, and separated each exposed group from site B. The main distinguishing index of each population selected by classification and regression tree analysis was MT-SH.     

Zn,Cu and Co in cyanobacteria: selective control of metal availability

Homeostatic systems for essential and non-essential metals create the cellular environments in which the correct metals are acquired by metalloproteins while the incorrect ones are somehow avoided. Cyanobacteria have metal requirements often absent from other bacteria; copper in thylakoidal plastocyanin, zinc in carboxysomal carbonic anhydrase, cobalt in cobalamin but magnesium in chlorophyll, molybdenum in heterocystous nitrogenase, manganese in thylakoidal water-splitting oxygen-evolving complex. This article reviews: an intracellular trafficking pathway for inward copper supply, the sequestration of surplus zinc by metallothionein (also present in other bacteria) and the detection and export of excess cobalt. We consider the influence of homeostatic proteins on selective metal availability.     

A new metallothionein gene from the giant keyhole limpet Megathura crenulata

Metallothioneins (MTs) are small soluble proteins ubiquitously expressed in animals and plants. Different isoforms are present in deuterostomes and protostomes. They do not differ greatly in primary structure, but are clearly distinguishable. Here, I present the gene and the complete cDNA of a novel MT from the mollusk Megathura crenulata. This protein is closely related to the Cu-inducible MTs of the vineyard snail Helix pomatia, but has also some minor sequence features typical of Cd-inducible isoforms of H. pomatia and other molluscs. Overall, the deduced primary structure is similar to the known molluscan MTs, but in addition possesses an insertion of 5 amino acids not found in any other molluscan MTs, protostomic or deuterostomic MTs. In addition, a pentapeptide insertion, characteristic of mammalian MT-3 is present but it lacks the functional tetrapeptide CPCP within the beta-region of those MT-3 proteins that are known to suppress neuronal growth processes. The M. crenulata MT is a novel form of MT in comparison to all other known MTs. Possible functional aspects for this new MT are discussed.     

Hepatic metallothioneins in two neotenic salamanders,Proteus anguinus and Necturus maculosus (Amphibia,Caudata)

The presence of metallothionein (MT) and the subcellular distribution of copper, zinc and cadmium were investigated in livers of two neotenic salamanders, Proteus anguinus and Necturus maculosus. In P. anguinus, caught in the wild, hepatic MTs were present as a single isoform of (Zn, Cu, Cd)-thioneins, whose molecular weight was estimated to be approximately 12000 by size exclusion chromatography. The percentage of zinc and cadmium was higher in the cytosol and of copper in the pellet. Cytosolic cadmium was almost exclusively associated with MTs (80%), while zinc and copper were also present in the regions of higher-molecular weight proteins. In laboratory bred N. maculosus, MTs were isolated from the liver cytosol and extract of the pellet as (Cu, Zn)- and (Zn, Cu)-thioneins, respectively. According to the low amount of copper extracting from liver pellets of N. maculosus, the presence of water insoluble aggregated forms of Cu-thioneins should be checked in further investigations.     

Metals in squid,Loligo forbesi,adults, eggs and hatchlings. No evidence for a role for Cu- or Zn-metallothionein

An adult squid Loligo forbesi had the following metals in its liver/digestive gland: Mn, Fe, Ni, Zn, Cu, As, Cd, Ba and Pb in the range of 1-110 ppm wet wt. Adult mantle muscle, adult eyes, eggs and hatchlings contained a lesser number of these metals at concentrations above 1 ppm. Chromatographic analysis of non-heat-treated cytosols (in the presence of 5 mM 2-mercaptoethanol) gave no evidence for the presence of copper- or zinc-containing fractions with the molecular weights of mollusc metallothioneins in any of the above tissues. Copper and Zn were bound to either the particulate fraction or to very low molecular weight species.     

Metallothionein 2A interacts with the kinase domain of PKCmu in prostate cancer

Prostate cancer (PC) patients die from progression to androgen independence (AI) and chemoresistance (CR). Protein kinase Cmu (PKCmu) a novel member of the PKC family of signal transduction proteins is downregulated in AI PC. Studying PKCmu interactors in the yeast two-hybrid system identified metallothionein 2A (MT 2A) as an interactor of PKCmu kinase domain (KD) in PC, which was quantified by beta-gal assay, confirmed in PC cells by immunoprecipitation, and PKCmu-MT 2A co-localization in vivo by immunofluorescence studies. PKCmu domain interaction studies revealed that MT 2A interacted strongly with KD, relatively weakly with C1, and failed to interact with C2, PH or kinase mutant domains. Peptide library and in silico analysis strongly suggest that MT 2A is a novel substrate of PKCmu and our data indicate that the PKCmu-MT 2A interaction depends on PKCmu kinase activity. Because metallothioneins are associated with cell proliferation and CR, the PKCmu-MT 2A interaction may contribute to CR and/or AI in PC.