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.

晚期非小细胞肺癌组织中ERCC-1、Metallothionein、p53表达与铂类耐药性及预后的相关性分析

背景与目的晚期非小细胞肺癌含顺铂方案化疗敏感性分子预测指标尚处于探索阶段。本研究分析晚期非小细胞肺癌石蜡包埋组织中错配切除修复蛋白(excisionrepaircrosscomplement-1,ERCC-1)、金属硫蛋白(metallothionein,MT)、p53蛋白与顺铂化疗敏感性的关系,并观察这些指标与病人生存的关系。方法收集1994年1月-2001年12月在本院经病理组织学诊断的用含铂方案(Gemzar+DDP或NVB+DDP)化疗的初治晚期非小细胞肺癌患者的病理标本,采用免疫组化方法检测ERCC-1、MT、p53的表达,并将病人的疗效和生存资料与上述指标的表达进行比较。样本率的比较用χ2检验,用Kaplan-meier法和Cox回归进行生存分析。结果本组51例标本中,ERCC-1阳性率为42.8%,MT阳性率为57.5%,p53阳性率为37.8%。化疗有效率在ERCC-1、MT、p53阴性组和阳性组分别为33.3%、35.3%、21.4%和16.7%、27.3%、35.3%(即分别为1.99倍,1.29倍,0.61倍),但均未达统计学差异(P>0.05),ERCC-1,MT均阴性组有效率为37.5%,均阳性组有效率为14.3%,前者为后者的2.6倍,但未达统计学差异(P>0.05)。平均生存期ERCC-1、MT、p53蛋白阴性组和阳性组分别为621.03天/523.14天,556天/479天,599.64天/416.60天。Kaplan-meier分析显示三者过度表达均提示预后不良,但Cox回归分析显示只?

Metallothionein:An overview

Metallothioneins （MTs） were discovered in 1957 by Margoshes and Vallee and identified as low-molecular weight and sulphydryl rich proteins. It is not surprising that most mammalian tissues contain age related basal levels of MTs since they are involved in metalloregulatory processes that include cell growth and multiplication. In an effort to understand the biology of this intriguing tumor, various biomarkers such as oncogenes, p53 tumor suppressor gene, war 1 protein, proliferating cell nuclear antigen, telomerase, microsatellite markers and cytogenetic changes have been examined. One biomarker which has recently shown to be expressed in various human tumors but still less reported in carcinoma is MT. Immunohistochemical detection of MT proteins in cold acetone-fixed paraffin embedded liver sections was performed by the streptavidin-avidin-biotin immunoperoxidase complex method.

小鼠肝脏金属硫蛋白Metallothioneins的分离纯化与鉴定

由健康小鼠通过皮下注射一定量的氯化镉,取肝脏匀浆,离心,经SephadexG-50和DEAE-Sephadex A-50柱层析分离,即可获得MT-Ⅰ和MT-Ⅱ两种金属硫蛋白。经鉴定:两个分子各含18个巯基,结合4个Cd原子和3个Zn原子,无金属蛋白质Thionein的分子量约为6kD,半胱氨酸含量约占氨基酸总量的28％,所提取到的MT-Ⅰ和MT-Ⅱ经氨基酸组成,HPLC和PAGE分析皆证明为高度均一的,且与有关文献报道基本相符。其中MT-Ⅱ已获得晶体。

p53 negativity, CDC25B positivity, and metallothionein negativity are predictors of a response of esophageal squamous cell carcinoma to chemoradiotherapy

AIM： Esophageal squamous cell carcinoma is generally sensitive to chemoradiotherapy （CRT）, but some cases are not. Using a retrospective analysis, we aimed to identify the predictors of the response by esophageal squamous cell carcinoma to definitive CRT. METHODS： The intensities of expression of p53, Ki67, Bci-2, Bax, olclin D1, VEGF, CDC25B, and metallothionein （MT） were evaluated immunohistochemically in the biopsy specimens obtained before CRT, and the intensities of their expression were tested for correlations with the clinical effects of CRT. RESULTS： The esophageal squamous cell carcinomas with negative p53, positive CDC25B, and negative MT expression were found to be significantly more sensitive to CRT. In addition, p53 positivity and CDC25B positivity respomd well to CRT. CONCLUSION： Esophageal squamous cell carcinomas with negative p53,positive CDC25B, and negative MT expressions respond well to CRT. Even with p53 positivity, if with CDC25B positivity, CRT can be expected. 2005 The WJG Press and Elsevier Inc. All rights reserved

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.

金属硫蛋白(Metallothionein,MT)基因在转基因领域的应用

MT丰富的巯基含量及重金属结合能力决定了它功能方面的多样性。本文综述了其高效可诱导性的启动子及其富含半胱氨酸的结构基因在细胞分子生物学 ,医学 ,农业及环保方面的转基因的应用及其发展过程。并对其在疾病诊断 ,环境保护等方面的前景进行了探讨。

Protective role of metallothionein in stress-induced gastric ulcer in rats

AIM: To illustrate the pathophysiological role of metallothionein (MT) in gastric ulcer induced by stress. METHODS: Wistar rats underwent water-immersionrestraint (WIR) stress, ZnSO4 (an MT inducer) treatment, WIR+ZnSO4 or WIR+MT, and the ulcer index (UI) was estimated in excised stomach and liver tissues. The mRNA level of gastric MT was determined by semi-quantitative RT-PCR. The MT content in gastric and hepatic tissues was determined by Cd/hemoglobin affinity assay. The lipid peroxidation products malondialdehyde (MDA) and conjugated dienes (CD) were estimated by use of thiobarbituric acid reactive species and ultraviolet spectrophotometry. RESULTS: WIR stress induced severe gastric mucosal lesions in rats. Compared with control rats, stressed rats had increased lipid peroxide content in serum and stomach and liver tissues. MDA content was increased by 34%, 21% and 29% and CD level by 270%, 83% and 28%, respectively. MT content in the stomach and liver was increased by 0.74- and 1.8-fold, and the MT-mRNA level in the stomach was increased by 26%. Pretreatment with ZnSO4 prevented gastric lesion development (the UI was 87% lower than that without pretreatment), and the MDA and CD content in serum and tissues was lower. The MT content in the liver was double in rats that were not pretreated, and the MT mRNA level in the stomach was 35% higher. MT administration 1 h before the WIR stress prevented gastric lesion development (the UI decreased by 47% compared with that in rats not pretreated), and the MDA and CD content in serum and tissues was significantly lower. CONCLUSION: In WIR-stressed rats, the MT level was increased in serum and in stomach and liver tissues. Pre-administration of exogenous MT or pre-induction of endogenous MT can protect the gastric mucosa against stress-induced ulcers and inhibits the formation of stressinduced lipid peroxide. MT could have a gastroprotective effect and might be a new interventive and therapeutic target in stress-induced gastric ulcers.

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

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.

Role of MetallothioneinlH in Cisplatin Resistance of Non-Small Cell Lung Cancer Cells

Objective： Despite platinum-based adjuvant chemotherapy has improved greatly patients＇ outcomes, drug resistance poses a major impediment to the successful use of such an effective agent. Metallothioneins（MTs） are known to play putative roles in cancer cell proliferation, apoptosis, differentiation, drug resistance and prognosis. The present studiy was to investigte the role of metallethioeinlH（MTIH） in cisplatin resistance of human non-small cell lung cancer（NSCLC） cell lines in vitro or its possible molecular mechanisms. Methods： MTIH mRNA expression in A549 and A549/DDP cells was detected by RT-PCR. A recombinant eukaryotic expression plasmid pcDNA3.1（-）-MT1H was constructed and transfected into A549 cells which express no MTIH. MT1H siRNA was transfected into A549/DDP cells which express MTIH highly. MTIH expression was detected by RT-PCR and Immunoblot. The chemosensitivity to cisplatin was assessed by MTT assay. Apoptosis rate was determined by Tunel and FCM. Bcl-2 and Bax were determined by immunohistochemistry. Results： MT1H mRNA was expressed in A549/DDP but not in A549. After transfection of MT1H, MT1H expression was enhanced and the chemosensitivity to cisplatin was decreased in A549 cells. Inversely, after transfection of MT1H siRNA, MT1H expression was decreased and the chemosensitivity to cisplatin was increased in A549/DDP. The apoptosis rate induced by cisplatin was increased and Bcl-2 was down-regulated but Bax showed little change in A549/DDP cells interferred with MT1H siRNA. Conclusion： MT1H overexpression can promote drug resistance in A549 cells . Down-regulation of MTIH interfered with siRNA can effectively reverses the drug resistance in A549/DDP cells by down-regulating the expression of Bcl-2 and increasing cisplatin induced apoptosis. SiRNA targeting MT1H combined with chemotherapy may be a very promising strategy for treatment of lung cancer.

Metallothionein 1 Isoform Gene Expression Induced by Cadmium in Human Peripheral Blood Lymphocytes

Objective To study the gene expression of metallothionein 1 （MT-1） isoforms in human peripheral blood lymphocytes （HPBLs）. Methods The expression of mRNA representing the seven active MT-I genes was determined in HPBLs by quantitative RT-PCR before and after exposure to cadmium. Results Basal expressions of MT-IX, and MT-1A in HPBLs were similar to expression of housekeeping gene. In contrast, the basal gene expressions of MT- 1 H, IF, 1E, and 1G were a little transcripts in human HPBLs. No signal was detected for MT-1B. There was a sex difference （P〈0.05）. in basal gene expression of MT-1E. The levels of gene expression of MT-1A, 1E, IF, 1G, 1H, and 1X increased, but the level of MT-1B did not increase after exposure to cadmium. Conclusions Gene expressions of MT-1 G, MT-1 H, MT-1 F, and MT-1X in HPBLs can be used as a potential biomarker of cadmium exposure.

Effects of Metallothionein on Isolated Rat Heart

To investigate the effects of metallothionein （MT） on isolated rat heart, 16 Wistar rats were randomly divided into 2 groups. In control group （group C）, distilled water was injected intraperitoneally and 24 h later isolated hearts were perfused with Langendorff and stored at 4℃ for 3 h with histidine-tryptophan-ketoglutarate （HTK） solutions, and then isolated hearts were perfused for 2 h by Langendorff. In experimental group （group E）, 3.6% ZnSO4 was injected intraperitoneally, 24 h later isolated hearts were perfused by Langendorff and stored at 4℃ for 3 h with HTK solutions, and then the isolated hearts were perfused for 2 h with Langendorff. MT content, the recovery of hemodynamics, myocardial water content （MWC）, lactate dehydrogenase （LDH） and creatine kinase （CK） leakage, adenosine triphosphate （ATP） and malondialdehyde （MDA） content, superoxide dismutase （SOD） activity, myocardial cell Ca^2＋ content, Ca^2＋-ATPase activity of mitochondria （[Ca^2＋-ATPase]m） and its Ca^2＋ content （[Ca^2＋]m）, synthesizing ATP activity of mitochondria （[ATP]m）, and the ultrastructure of cells were examined. There were a significant increase in group E in hemodynamic recovery, ATP content, SOD activity, [Ca^2＋-ATPase]m activity, [ATP]m activity, and substantial reduction in MWC, LDH and CK leakage, MDA content, myocardial cell Ca^2＋ content, [Ca^2＋]m content, and the ultrastructural injury were obviously milder than that of group C. This study demonstrated that MT has protective effects on isolated rat heart.

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.

Focusing on the protective effects of metallothionein-Ⅰ/Ⅱ in cerebral ischemia

Ischemic stroke is a leading cause of death and has a remarkable social and economical impact, which rises with the increasing age of the industrial population. Unfortunately, treatment strat- egies for cerebral ischemia still remain very limited. Acute reper- fusion therapies with either systemic thrombolysis using rt-PA （recombinant tissue plasminogen activator） or interventional recanalization procedures were shown to be highly effective. In addition, there is also a long-existing concept to modulate stroke-associated pathophysiological events such as exitotox- icity, peri-infarct depolarizations, apoptosis and inflammation （Dirnagl et al., 1999）.

Metallothionein克隆载体及表达载体的构建及转化矮牵牛的初步研究

Metallothionein基因位于文库克隆载体pTriplEx2上,活化菌液并用限制性内切酶SfiI酶切,同时酶切添加了SfiI酶切位点的环状pMD-18TM,回收目的片段和载体并连接,经过酶切和PCR验证,成功构建克隆载体;再用XbaI和SmaI分别双酶切克隆载体和表达载体pc2301,回收目的片段和表达载体并连接,经酶切和PCR验证,成功构建含有目的基因的表达载体,进而转化植物获得耐重金属的植物株系。初步验证转化后的植株具有抗击重金属的能力。

Metallothionein Genes in the Nematode Caenorhabditis elegans and Metal Inducibility in Mammalian Culture Cells

Genomic DNAs of metallothionein Ⅰ and Ⅱ in Caenorhabditis elegans (CeMT-Ⅰand CeMT-Ⅱ) were isolated by YAC library/polytene filter hybridization followed by subcloning of corresponding cosmid clones. Both genes are mapped at chromosome V. Although the similarities of 5'-flanking regions and coding regions have shown only 55-58%, the introns are split at the same position in both genes, indicating that these two genes are originally from the same gene. While several metal responsive elements are conserved among eukaryotes, only one metal responsive element was found in the promoter region in CeMT-Ⅱ and not in CeMT-Ⅰ. Indced, neither of 5'-flanking regions of CeMT-Ⅰ nor CeMT-Ⅱ connected to chloramphenicol acetyltransferase reporter gene is responsive to heavy metals in mammalian culture cells by transient transfection analysis. These results would suggest that the metal regulatory factors in C.elegans might be different from those conserved in invertebrates and vertebrates, although the MTs in C elegans revealed the similarities to mammalian MTs in several points

THE INDUCTION AND EXTRACTION OF METALLOTHIONEINS IN ARTEMIA

A small amount of heavy metal binding protein,identified by BioRad Protein Assay,has been iso-lated from the adult brine shrimp,Artemia franciscanus.This protein has an apparent molecular weight be-tween 6000 to 9000 dalton.a UV absorption peak at 260 instead of 280 nm like most proteins;andhas high affinity towards binding with radioactive labeled 109Cd.These characteristics are similarto that of metallothioneins reported for many vertebrate and invertebrate,marine and terrestrialanimals.After the brine shrimp is exposed to a small amount of Cd2+for 24 h,a large amount ofmetallothionein can be isolated,showing the inducibility of this detoxifying protein in the adult Artemiain a short period of time.

Quantitation of metallothionein and cadmium in metallothionein in mink livers by anion-exchange HPLC-GFAAS method

Metallothionein (MT) has a great capacity of binding heavy metals showing an interesting connection with metal toxicology, as a biochemical marker for environmental metal pollution. Anino-exchange high per formance liquid chromatography (HPLC) was used to isolate and quantitate MT in livers of minks which were contaminated with heavy metals. MT isoforms (MT-I and MT-II) were eluted at approximately 11.3 and 14.3 min respectively from a DEAE-5 PW anion-exchange column with a Tris-HCl buffer (0.01 -0.25 mol/L, pH 8.6) and detected by UV absorbance at 254 nm. The cadmium concentrations in mink liver MT elutkms were determined by graphite furnace atomic absorption spectrometry (GFAAS) . Obvious increase in liver MT-I concentration rather than liver MT-II was found when the minks were contaminated by feeding contaminated fish captured from the heavy metal-polluted river. The cadmium concentration in mink liver MT-I also increased to some extent as the contaminated level increased.