金属硫蛋白基因工程菌对Cd的去除研究

研究了金属硫蛋白基因工程菌对温度的耐受性,以及在一定温度条件下对Cd的去除率。通过10～50℃温度范围内的静态实验,表明枣金属硫蛋白基因工程菌的温度耐受范围为10～50℃,在工程菌的OD值能维持在2左右时,表明金属硫蛋白基因工程菌的最佳温度范围为25～45℃。通过陶瓷和活性炭两种填料的动态实验,考察了在不同温度条件下基因工程菌对Cd的富集情况,表明在陶瓷填料中基因工程菌下对Cd的去除率达到98.5%。

ZNT1 Involves Cuproptosis through Regulating MTF1-conduced Expression of MT1X under Copper Overload

Industrial activities such as smelting emissions,mineral combustion and industrial wastewater discharge might lead to copper pollution in the environment.This kind of copper pollution has harmful effects on aquatic o rganisms,plants and animals through direct or indirect exposure.However,the current understanding of the toxicity of copper is rather limited.Copper overload can perturb intracellular homeostasis and induce oxidative stress and e ven cell death.Recently,cuproptosis has been identified as a copper-dependent form of cell death induced by o xidative stress in mitochondria.We uncover here that zinc transporter 1(ZNT1)is an important regulator involved in cuproptosis.Firstly,we established the copper overload-induced cell death model with the overexpression of copper importer SLC31A1 in HeLa cells.Using this model,we conducted unbiased genome-wide CRISPR-Cas9 screens in cells treated with copper.Our results revealed a significant enrichment of ZNT1 gene in both library A and library B plasmids.Knocking out of ZNT1 in HeLa cells notably prevented cuproptosis.Subsequent knockout of metal transcription factor 1(MTF1)in ZNT1-deficient cells nearly abolished their ability to resist copper-induced cell death.However,overexpression of metallothionein 1X(MT1X)in the double-knockout cells could p artially restored the resistance to cuproptosis by loss of MTF1.Mechanistically,knockout of ZNT1 could promote MT1X expression by activating MTF1.As a consequence,the interaction between MT1X and copper was e nhanced,reducing the flow of copper into mitochondria and eliminating mitochondria damage.Taken together,this study reveals the important role of ZNT1 in cuproptosis and shows MTF1-MT1X axis mediated resistance to c uproptosis.Moreover,our study will help to understand the regulatory mechanism of cellular and systemic copper homeostasis under copper overload,and present insights into novel treatments for damages caused by both genetic copper overload diseases and environmental copper contamination.

Cloning and Expression Patterns of a Metallothionein-like GenehtMT2 of Helianthus tuberosus

A novel cDNA sequencehtMT2, which encodes a type 2 metallothionein_like protein, was isolated from Helianthus tuberosus L. tuber cDNA library. The whole sequence is 509 bp, including an open reading frame (ORF) of 240 bp, a 5′ UTR of 62 bp and a 3′ UTR of 207 bp. Two genomic sequences covering the coding region ofhtMT2were cloned by PCR reaction. Sequence analysis revealed that the genomic sequences htMTG_1 of 986 bp and htMTG_2 of 982 bp were both composed of three exons and two introns. The deduced protein consisted of 79 amino acid residues with a predicted molecular weight of 7.8 ku (kD). Amino_terminal and carboxy_terminal domains contained 8 and 7 cysteine residues respectively, separated by a central cysteine free spacer. Sequence alignment revealed that the predicted protein ofhtMT2 was homologous to type 2 metallothioneins (MTs) of plants. Southern blotting analysis indicated that htMT2was encoded by a small multi_gene family in H. tuberosus genome. Northern blotting analysis showed that htMT2 transcripts were detected in stems, leaves and leafstalks, but no transcripts were detected in roots. The expression level in stems was the highest among the above tissues. Transcripts in stems were significantly reduced by Cu 2+ treatment. Judging from the homologies between the deduced HtMT2 and other type 2 plant metallothioneins as well as responses to metal ions, we believe thatwere cloned by PCR reaction. Sequence analysis revealed that the genomic sequences htMTG_1 of 986 bp and htMTG_2 of 982 bp were both composed of three exons and two introns. The deduced protein consisted of 79 amino acid residues with a predicted molecular weight of 7.8 ku (kD). Amino_terminal and carboxy_terminal domains contained 8 and 7 cysteine residues respectively, separated by a central cysteine free spacer. Sequence alignment revealed that the predicted protein ofhtMT2 was homologous to type 2 metallothioneins (MTs) of plants. Southern blotting analysis indicated that htMT2was encoded by a small multi_gene family in H. tuberosus genome. Northern blotting analysis showed that htMT2 transcripts were detected in stems, leaves and leafstalks, but no transcripts were detected in roots. The expression level in stems was the highest among the above tissues. Transcripts in stems were significantly reduced by Cu 2+ treatment. Judging from the homologies between the deduced HtMT2 and other type 2 plant metallothioneins as well as responses to metal ions, we believe that[ShtMT2 encodes a new type 2 metallothionein.

Modeling Rice rgMT as a Plant Metallothionein-Like Protein by the Distance Geometry and Homology Methods

Rice metallothionein-like protein (rgMT) shows characteristics of a three-section pattern composed of two highly conserved cysteine rich (CR) domains in the terminals and a spacer without cysteine (cys) residues in the center of the molecule. In this paper, the two CR domains and the spacer region were modeled by the distance geometry and homology methods separately. For the CR domains, twenty random models were generated for each cys combination based on the constraint conditions of CXC (C represents cys, X represents any amino acid other than cys), and CXXC motifs and a metal-sulfur chelating cluster. Four models for the N-terminal and two for C-terminal CR domain containing metal chelating structures formed by different combinations of cys were selected from 900 possible conformations. The GOR method was used to predict the secondary structure of the spacer region and its model was built by the homology method. After three parts of the protein were modeled, they were connected to form a three-dimensional structure model of rgMT. The whole conformation showed that rgMT could form two independent metal-sulfur chelating structures connected by a spacer peptide, without a structural or energy barrier for them to form two independent metal-chelating clusters just as mammalian metallothionein (MT) proteins. As all plant metallothionein-like (MT-L) proteins have the same primary structural characteristic, two CR domains connected by a spacer region, and many have the same cys arrangement pattern as rgMT, the three-dimensional structure model of rgMT will provide an important reference for the structural study of other plant MT-L proteins.

Bioinformatics Analysis of Metallothionein in Betula phatyphylla

[Objective] This study aimed to analyze the bioinformatics information of Betula phatyphylla metallothionein for subsequent investigation of the important func- tions of metallothionein gene. [Method] The physical and chemical properties, trans- membrane domains, hydrophobicity / hydrophilicity, secondary structure, functional domains and functional classification of B. phatyphylla metallothionein were analyzed and predicted using bioinformatics methods and tools. [Result] The relative molecular mass of B. phatyphylla metallothionein is 7.75 kD and isoelectric point is 5.66; there is a strong hydrophobic region in the center of B. phatyphylla metallothionein and hydrophilic regions in both ends; B. phatyphylla metallothionein may be a non-trans- membrane protein and is mainly constituted by random coil without enzymatic activi- ty, suggesting that it belongs to Metallothio_2 protein family. [Conclusion] This study provided reference for further functional research of B. phatyphylla metallothionein gene.

Research Progress on Heavy Metals Detoxification in Human Body

With the pollution of heavy metals becoming more and more serious, hu- man health suffers from great harms, making the detoxification in human body admit of no delay. This paper summarized the heavy metals used for detoxification in hu- man body in recent years, including metallothionein, polyphenols, dietary fibers from seaweed, GTS, and explored the detoxification mechanism of these heavy metals in human body.

Modeling the Cysteine Rich Domain of Plant Metallothionein-like Protein

With the progress of plant genome research, more than 50 plant metallothionein_like (MT_L) genes have been found, but only several MT_L proteins have been detected and no experimental structural information for MT_L proteins has been reported so far. Since detailed knowledge of the protein tertiary structure is required to understand its biological function, a method is needed to determine the structure of these proteins. In this study, the structural data of known mammal MT was used to determine the interatomic distance constraints of the CXC and CXXC motifs and the metal_sulfur chelating cluster. Then several possible MT conformations were predicted using a distance geometry algorithm. The statistical analysis was used to select those with much lower target function values and lower conformation energies as the predicted tertiary structural models of the cysteine_rich (CR) domains of these proteins. A suitable prediction method for modeling the CR domain of the plant MT_L protein was constructed. The accurately predicted result for the known structure of an MT protein from blue crab suggests that this method is practicable. The tertiary structures of CR domains of rape MT_L protein LSC54 was then modeled with this method.

Inhibition of cysteine protease papain by metal ions and polysulfide complexes,especially mercuric ion

Aim Cysteine proteases are closely associated with many human and non-human pathological processes and are potential targets for metal ions especially Hg^2＋ and the related species. In the present work, on the basis of to the general study on the effects of some metal ions on the activity of papain, a well-known representative of cysteine protease family, the inhibitory effects of Hg^2＋ and polysulfide complexes were studied. Results All the metal ions tested （Hg^2＋, Cu^2＋, Ag^＋, Au^3＋, Zn^2＋, Cd^2＋, Fe^3＋, Mn^2＋, Pb^2＋, Yb^3＋） inhibit the activity of papain anda good correlation between the inhibitory potency and softness-and-hardness was observed. Among the metals, Hg^2＋ was shown to be a potent inhibitor of papain with a Kiof 2 × 10^-7 mol·L^-1 among. Excessive amounts of glutathione and cysteine could reactivate the enzyme activity of papain deactivated by Hg^2＋. These evidences supported that Hg^2＋ might bind to the catalytic site of papain. Interestingly, Hg （Ⅱ） polysulfide complexes were for the first time found to inhibit papain with a Ki of 7 × 10^-6 mol·L^-1, whose potency is close to a well known mercury compound, thimerosal （Ki=2.7 × 10^-6）. In addition, Hg （Ⅱ） polysulfide complexes exhibit good permeability （ 1.9 × 10-5 cm· s^-1） to caco-2 monolayer. Conclusion These results suggested that mercury polysulfide complexes might be potential bioactive species in the interaction with cysteine proteases and other- SH-content proteins, providing a new clue to understand the mechanism of the toxicological and pharmacological actions of cinnabar and other insoluble mercury compounds.

Clinical signification on the expressions of metallothionein in three types cancer of woman

Objective： To study the expressions and significations of metallothionein （MT） in cervical squamous cell cancer （CSC）, bladder transitional cell cancer （BTC） and breast cancer （BC） of woman. Methods： Immunohistochemical method was used to examine the expresses rate of MT in three types of woman cancer tissue. Results： The expressions rates of MT were 54.35% （29146） in BTC, 67.05% （59188） in BC and 57.14% （40/70） in CSC. The positive rate of MT expression was higher in low differentiation group than well differentiation group in BTC and CSC （P 〈 0.05）. Positive of MT in lobular cancer was significance higher than medullary and duct cancers （P 〈 0.05）. Conclusion： The expression of MT is related to differentiation degree, and it is a guidance for clinical choice of chemotherapy project.

p53 antibodies,metallothioneins,and oxidative stress markers in chronic ulcerative colitis with dysplasia

AIM:To investigate the role of p53 antibodies (p53Abs),metallothioneins (MTs) and oxidative stress markers in the early detection of dysplasia in chronic ulcerative colitis (UC).METHODS:The study included 30 UC patients,15 without dysplasia (group Ⅱ) and 15 with dysplasia (group Ⅲ),in addition to 15 healthy volunteers (group Ⅰ,control subjects).The enzyme-linked immunosorbent assay technique was used to measure serum p53Abs and MTs,while advanced oxidation protein products (AOPPs),and reduced glutathione (GSH) levels were measured by spectrophotometric method in all subjects.RESULTS:In group Ⅱ and group Ⅲ compared to group Ⅰ,there were significant increases in serum levels of AOPPs (145.94 ± 29.86 μmol/L and 192.21 ± 46.71 μmol/L vs 128.95 ± 3.06 μmol/L,P < 0.002 and P <0.001,respectively),MTs (8.18 ± 0.35 μg/mL and 9.20 ± 0.58 μg/mL vs 6.12 ± 0.25 μg/mL,P < 0.05 and P < 0.05,respectively),and p53Abs (20.19 ± 3.20 U/mL and 34.66 ± 1.34 U/mL vs 9.42 ± 1.64 U/mL,P < 0.001 and P < 0.001,respectively).There were significantly higher levels of AOPPs (P < 0.05) and p53Abs (P < 0.001) in UC patients with dysplasia compared to those without dysplasia,while MTs showed no significant difference between the 2 groups (P > 0.096).In contrast,GSH levels showed a significant decrease in both patients' groups (1.87 ± 0.02 μmol/mL and 1.37 ± 0.09 μmol/mL vs 2.49 ± 0.10 μmol/mL,P < 0.05 and P < 0.05 in groups Ⅱ and Ⅲ,respectively) compared with group Ⅰ,and the levels were significantly lower in group Ⅲ than group Ⅱ (P < 0.05).There was a positive correlation between AOPPs and both MTs (r=0.678,P < 0.001) and p53Abs (r=0.547,P < 0.001),and also between p53Abs and MTs (r=0.739,P < 0.001).There was a negative correlation between AOPPs and GSH (r =-0.385,P < 0.001),and also between GSH and both MTs (r=-0.662,P < 0.001) and p53Abs (r=-0.923,P < 0.001).CONCLUSION:Oxidative stress and oxidative cellular damage play an important role in the pathogenesis of chronic UC and the associated carcinogenetic process.p53Abs levels could help in early detection of dysplasia in these conditions.

Expression of Mouse MT-1 as a Fusion Protein in Anabaena sp. PCC 7120

To produce mouse metallothionein-1 (mMT-I ) in cyanobacterium Anabaena sp. PCC 7120, a novel Escherichia co/i-cyanobacterium shuttle fusion expression vector, pKG-MT, was constructed. Via this vector, mMT-I cDNA which was fused with a carboxyl terminal extension of the 26 kD glutathione-S-trans-ferase (GST) containing a thrombin specific site was expressed in Anabaena under the control of tac promoter. SDS-polyacrylamid gel electrophoresis (SDS-PAGE) showed that the fusion protein GST-MT was expressed in the transgenic Anabaena sp. PCC 7120 after induction with isopropylthio-β-D-galactoside (IPTG). Glutatione-S-transferase metallothionein (GST-MT) was purified from the crude extracts by affinity chromatography on immobilized glutathione and mMT- I was obtained by digesting the fusion protein with thrombin on column and gel filtration on Sephadex G-50. SDS-PAGE demonstrated that the purified mMT- I was the desired protein. The result of ELISA for the purified mMT-I showed that the recovery of mMT- I from the transgenic cyanobacterium was about 0.6 mg/g fresh weight. According to the data of atomic absorption assay, metal-binding activity of the purified mMT-I was almost the same as that of wild type MT.

Dietary zinc and metallothionein on small intestinal disaccharidases activity in mice

AIM: To examine the effect of increasing dietary zinc (Zn) intake and the lack of metallothionein (MT) expression on activity of small intestinal disaccharidases. METHODS: MT-Ⅰ and Ⅱ knockout (MT-/-) and wild-type (MT+/+) female mice at 3.5 wk of age were randomly fed with a diet containing 2 (2 Zn), 15 (15 Zn) or 50 (50 Zn) mg Zn/kg (n = 8/group/genotype) for 5 wk. Small intestinal segments (duodenum, jejunum and ileum) were collected and either fixed in 10% formalin for histological analysis or snap frozen in liquid nitrogen for sucrase, lactase and maltase activity analyses.RESULTS: Plasma Zn was significantly (P < 0.05) lower (33%) in MT-/- compared with MT+/+ mice fed the 2 Zn diet. Villus height and crypt depth were increased by approximately 15% in MT+/+ mice compared with MT-/- mice. Duodenal disaccharidase activities were significantly higher in MT+/+ compared with MT-/- mice particularly in those fed the 2 Zn diet. For the 50 Zn diet, jejunal sucrase and lactase activities were significantly higher in MT-/- (13 313 ± 2314; 4107 ± 364 μmol glucose/well/min/g tissue, respectively) compared with MT+/+ mice (7054 ± 608; 1818 ± 174). Similarly, ileal lactase activities were higher in MT-/- (1480 ± 192) compared with MT+/+ (629 ± 353) mice particularly those fed the 2 Zn diet. CONCLUSION: Increasing dietary Zn has little effect on disaccharidases activity in MT wild-type mice. The presence of MT may enhance morphological and functional development of the gut.

A cadmium metallothionein gene of ridgetail white prawn Exopalaemon carinicauda(Holthuis,1950) and its expression

Metallothioneins （MTs） are a group of low molecular weight cysteine-rich proteins capable of binding heavy metal ions. A cadmium metallothionein （EcMT-Cd） cDNA with a 189 bp open reading frame （ORF） that encoded a 62 amino acid protein was obtained from Exopalaemon carinicauda. Seventeen cysteines were in the deduced amino acid sequence, and the cysteine （Cys）-rich characteristic was revealed in different metallothioneins in other species. In addition, the deduced amino acid sequence did not contain any aromatic amino acid residues, such as tyrosine （Tyr）, tryptophan （Trp）, and phenylalanine （Phe）. EcMT- Cd mRNA was expressed in all tested tissues （the ovary, muscle, stomach, and hepatopancreas）, and its expression profiles in the hepatopancreas were very different when shrimps were exposed to seawater containing either 50 pmol/L CuSO4 or 2.5 gmol/L CdC12. The expression of EcMT-Cd was significantly up-regulated in shrimp exposed to CuSO4 for 12 h and down-regulated in shrimps exposed to CdC12 for 12 h. After 24 h exposure to both metals, its expression was down-regulated. By contrast, at 48 h the EcMT-Cd was up-regulated in test shrimps exposed to CdC12. The transcript of EcMT-Cd was very low or even absent before the zoea stage, and the expression of EcMT-Cd was detected from mysis larvae-I, then its expression began to rise. In conclusion, a cadmium MT exists in E. carinicauda that is expressed in different tissues and during different developmental stages, and responds to the challenge with heavy metal ions, which provides a clue to understanding the function of cadmium MT

Effect of Metallothionein on Cell Cycle,Apoptosis Rate and Subsets Distribution of Lymphocytes in Peripheral Blood of Dairy Cattle under Heat Stress

[Objective] This study aimed to research the effect of metallothionein on cell cycle, apoptosis rate and subsets distribution of lymphocytes in peripheral blood of dairy cows under heat stress, so as to perfect the regulative mechanism re- searches of MT to anti-heat stress. [Method] Twenty lactating Chinese Holstein cows were randomly divided into four groups （A, B, C and D）, and injected with 0, 4.0, 8.0 and 12.0 mg Zn-metallothionein, respectively by intravenous route. Blood sam- ples were collected at 1＂, 16~, 31~, 46~ and 61~ day, and the dynamic changes of cell cycle, apoptosis rate and subsets distribution of lymphocytes were determined. [Result] The apoptosis rate of cells in group B and C was lower than those in group A by 26.63% （P〉0.05） and 24.84% （P〉0.05） respectively. The number of cells in the Gc/G1 phage in trial groups was increased and the number of cells in the S and GJM phages tended to decrease, but there were no significant differences （P〉 0.05）. The number of CD3~ T cell in three trial groups was greater than those in group A by 7.02% （P〉0.05）, 5.45% （P〉0.05） and 3.85% （P〉0.05） respectively, while the number of CD4~ T cell in trial groups was higher than those in control group by 31.04% （P〈0.05）, 35.68% （P〈0.05） and 39.34% （P〈0.05） respectively. The number of CD8＇ T cell and the levels of CD4＊/CD8~ in trial groups were increased observ- ably, but significant difference （P〈0.05） was observed in the levels of CD4~/CD8~ between groups A and C only. It demonstrated that exogenous Zn-metallothionein can decrease apoptosis rate, improve cell cycle and regulate subsets distribution of lymphocytes in dairy cattle in a dose-dependent manner. [Conclusion] This study will provide scientific basis for safe utilization of MT in dairy industry.

Concurrent repletion of iron and zinc reduces intestinal oxidative damage in iron- and zinc-deficient rats

AIM： To understand the interactions between iron and zinc during absorption in iron- and zinc-deficient rats, and their consequences on intestinal oxidant-antioxidant balance. METHODS： Twenty-four weanling Wistar-Kyoto rats fed an iron- and zinc-deficient diet （〈 6.5 mg Fe and 4.0 mg Zn/kg diet） for 4 wk were randomly divided into three groups （n = 8, each） and orally gavaged with 4 mg iron, 3.3 mg zinc, or 4 mg iron ＋ 3.3 mg zinc for 2 wk. At the last day of repletion, 3 h before the animals were sacrificed, they received either 37 mBq of SSFe or ^65Zn, to study their localization in the intestine, using microautoradiography. Hemoglobin, iron and zinc content in plasma and liver were measured as indicators of iron and zinc status. Duodenal sections were used for immunochemical staining of ferritin and metallothionein. Duodenal homogenates （mitochondrial and cytosolic fractions）, were used to assess aconitase activity, oxidative stress, functional integrity and the response of antioxidant enzymes. RESULTS： Concurrent repletion of iron- and zinc-deficient rats showed reduced localization of these minerals compared to rats that were teated with iron or zinc alone; these data provide evidence for antagonistic interactions. This resulted in reduced formation of lipid and protein oxidation products and better functional integrity of the intestinal mucosa. Further, combined repletion lowered iron-associated aconitase activity and ferritin expression, but significantly elevated metallothionein and glutathione levels in the intestinal mucosa. The mechanism of interactions during combined supplementation and its subsequent effects appeared to be due to through modulation of cytosolic aconitase, which in turn influenced the labile iron pool and metallothionein levels, and hence reduced intestinal oxidative damage.CONCLUSION： Concurrent administration of iron and zinc corrects iron and zinc deficiency, and also reduces the intestinal oxidative damage associated with iron supplementation.

Metallothioneins Induced by Antimony Accumulation in Earthworm Eisenia foetida Using Filter Paper Contact Method

MTs （Eiseniafoetida metallothioneins） have been recently reported as biomarkers for environmental metal contamination, however no study regarding Sb （antimony） is available. This study aimed to explore possibility of MTs as a biomarker of Sb pollution. A contact method of filter paper was used to investigate Sb inducing MTs in Eiseniafoetida. Spectrophotometrical and SDS-PAGE （sodium dodecyl sulphate-polyacrylamide gel electropheresis） analyses were used to determine the nature of the induced MTs samples. Rabbit liver metallothioneins was used as a standard. The results showed that the molecular weight of induced protein was approximately 6-7 kDa. A characteristic absorption peak at 268 nm was observed, which was consistent with the standard. The amounts of Sb-MTs quantified by cadmium hemoglobin saturation method showed a significant positive relationship with increasing Sb exposure.The experiments prove that Sb can induce Sb-MTs in Eiseniafoetida as a biomarker of antimony pollution.