Induction of Metallothionein in Rat Liver by Cadmium Chloride：Probable Mechanism of Action

The mechanism of action of cadmium in the inductive pathway of metallothionein(MT)synthesis was studied in the rat.Cadmium significantly elevated the MT level by 872% which was antagonised by coadministration of either verapamil(343.5%)or ionophore(570%).The Ca-dependent biomolecules such as cyclic AMP or calmodulin remained depressed in all treatment regimens except calmodulin in the ionophore treated rat.Total Ca^2 showed no increase in its profile except in the ionophore treatments either alone or with CdCl2.The Na^ profile is,however,significantly elevated in all cases except the ionophore treated rat.The present study clearly indicates that (a)Ca^2 has no first messenger role in the schematic events leading to MT synthesis and (b)Na^ may be regarded as a possible candidate in the molecular events of Cd-induced MT synthesis.     

EOLA1与MT2A相互作用及其上下游关系的确定

目的 研究内皮细胞高表达脂多糖相关因子1(EOLA1)与金属硫蛋白2A(MT2A)的相互作用关系,明确其相互作用位点及上下游关系.方法 构建EGFP-EOLA1融合表达载体,转染HUVEC细胞,应用免疫荧光共定位成像技术观察EOLA1和MT2A在HUVEC中的共定位情况;应用截短突变和酵母双杂交确定EOLA1和MT2A的相互作用位置;应用RNAi技术建立EOLA1/MT2A上调和下调的HUVEC细胞模型,观测模型细胞EOLA1/MT2A的表达情况.结果 EOLA1和MT2A在HUVEC中存在共定位现象,共定位主要发生在核膜周围;酵母双杂交和免疫共沉淀实验结果显示,EOLA1的截短突变体1～77氨基酸(aa)片段和MT2A无结合活性,1～115aa片段及1～158aa片段和MT2A有结合活性.EOLA1上调/下调表达时MT2A同时相应上调/下调表达,MT2A的上调和下调表达对EOLA1表达无明显影响.结论 EOLA1和MT2A在HUVEC细胞内存在相互作用,相互作用位点位于EOLA1ORF的100～113功能域,EOLA1可以调控MT2A在HUVEC中的表达.     

金属硫蛋白在宫颈癌中表达及其意义

目的 探讨金属硫蛋白(MT)在宫颈鳞状细胞癌中的表达状况。方法 对66例宫颈鳞状细胞癌(cSc)和30例宫颈上皮内瘤样增生(CIN)标本，进行免疫组化检测。结果表明 MT在CSC中阳性率为68．18％(45／66)，在CIN中阳性率为29．03％(8/30)，正常对照组阳性率为8．00％(2／25)。MT表达与癌组织分化程度有关。结论 MT在CSC中有比较高的表达，检测MT对化疗方案制定有指导意义。     

Influence of Isoflavones on Cadmium-induced Adverse Effects in Vascular Endothelial Cells （ECV 304）

To study the possible intervention of isoflavones in cytotoxicity induced by cadmium in vascular endothelial cells. Methods An ECV 304 cell line derived from human umbilical vein endothelial cells was adopted. Genistein / daidzein was added prior to or simultaneously with CdCl2, cell viability was determined by MTT assay, and metallothionein mRNA expression was monitored by RT-PCR method. Results Cell viability was higher in isoflavone and CdCl2. co-treated groups than that in CdCl2 treated group, with CdCl2 concentration at 10, 20, 40, and 80 μmol/L, respectively. However this increase was not observed in the group treated with CdCl2 at a concentration of 60 μmol/L, lsoflavones (10^-1mol/L to 10^-5 mol/L) were added 24 h before cells were challenged with 80 μmol/L CdCl2 for 24 h or simultaneously with 80 ^-10mol/L CdCl2. Genisteinincreased cell viability only at 10^-5 mol/L, while daidzein caused a dose-dependent increase from 10^-10 mol/L to 10^-5 mol/L in co-treatment with CdCl2. In pre-treatment, genistein (10^-7 to 10^-5 mol/L) increased cell viability whereas only 10^-5 mol/L of daidzein exerted protection. Apparent protection could be found when the cells were pre-treated with 10^-5 mol/L isoflavones for over 12 h, whereas 24 h incubation was required in such a co-treatment, with the exception of daidzein that had a significant protection in only 3 h. Isoflavones (10^-6 mol/L) incubated for 3 h to 24 h, increased MT ⅡA and MT IF mRNA expression, but the induction could not last for more than 24 h. Co-treatment with isoflavones could induce an additional induction of MT ⅡA mRNA expression in cells exposed to cadmium. However, the additional induction of MT ⅡA and MT IF mRNA was not seenwhen pre-treatment was carried out with isoflavones, with the exception of an increase in MT ⅡA mRNA expression in the daidzein pre-treated group. Conclusion Genistein/daidzein could reverse the cytotoxicity of cadmium either in pre-treatment or in co-treatment. The protection is the strongest in 10^-5 mol/L of isoflavones with a dose-dependent pattern. There are differences between genistein and daidzein in their protective effects. Whether the protection of isoflavones is related to their capacity of inducing MT mRNA expression remains to be elucidated.     

Expression of p57kip2 and cyclinE proteins in human pancreatic cancer

Objective To investigate the effects of p57kip2 and cyclinE proteins on the genesis and progression of human pancreatic cancer. Methods The expression of p57kip2 and cyclinE proteins in tumor tissues and adjacent tissues of pancreatic cancer in 32 patients was detected by SP immunohistochemical technique. Results The p57kip2 protein positive-expression rate in tumor tissues of pancreatic cancer was 46.9%, which was lower than that in adjacent pancreatic tissue (P<0.05). The p57kip2 protein positive-expression correlated significantly with tumor cell differentiation (P<0.05) and did not correlate significantly with lymph node metastasis (P>0.05). The cyclinE positive-expression rate in tumor tissues was 68.8%, which was higher than that in adjacent pancreatic tissues (P<0.05). The cyclinE positive-expression also correlated significantly with tumor cell differentiation and lymph node metastasis (P<0.05). The cyclinE protein positive-expression rate in the tumor tissues of the p57kip2 protein positive-expression group was lower than that in the p57kip2 protein negative-expression group, and there were no significant correlation between the two groups (r= -0.112, P＞0.05).Conclusion Decreased expression of the p57kip2 protein and/or over-expression of the cyclinE protein may play an important role in the genesis and progression of human pancreatic cancer.     

金属硫蛋白2A重组质粒的构建及其在血管内皮细胞中的表达

目的 建立人血管内皮细胞金属硫蛋白2A(MT2A)上调表达细胞模型.方法 构建重组质粒pEGFP-N1-MT2A,转染至人脐静脉内皮细胞(HUVECs),荧光显微镜观察转染效果,RT-PCR及蛋白印迹法测定细胞MT2A表达水平.结果 测序证实pEGFP-N1-MT2A重组质粒构建正确,转染至HUVECs后,通过RT-PCR蛋白印迹实验证实细胞MT2A表达水平升高.结论 成功构建了重组质粒pEGFP-N1-MT2A,转染至HUVECs可使MT2A表达水平升高.     

The Effect of TanshinoneⅡ A upon the TGF-betal/Smads Signaling Pathway in Hypertrophic Myocardium of Hypertensive Rats

To investigate the molecular mechanism by which Tanshinone Ⅱ A （TSN Ⅱ A） prevents left ventricular hypertrophy （LVH）, we examined the expression of AT1R, TGF-β1 and Smads gene in the hypertrophic myocardium of hypertensive rats with abdominal aorta constriction. LVH model was established by creating abdominal aorta constriction. Four weeks later, animals were randomly divided into 4 groups with 8 animals in each. One group was used as model control, the other three groups were treated with TSN ⅡA （20 mg/kg）, TSN ⅡA （10 mg/kg） and valsartan （10 mg/kg）, respectively. Another 8 SD rats were subjected to sham surgery and served as blank control. After 8- week treatment, the caudal artery pressure of the animals was measured. The tissues of left ventricle were taken for the measurement of the left ventricular mass index （LVMI） and pathological sectioning and HE-staining were used for determining the myocardial fiber dimension （MFD）. The mRNA expression of AT1R, protein expression of TGF-betal and activity of Smad-2, 4, 7 were detected by RT-PCR and Western blotting, respectively. Our results showed that （1） the blood pressure of rats treated with TSN Ⅱ A, either at high or low dose, was significantly higher than those in the control and valsartan-treated group （P〈0.01, P〈0.05）; （2） LVMI and MFD in TSN Ⅱ A and valsartan-treated rats were higher than those in the control group （P〈0.05） but significantly lower than those in the model control （P〈0.01）; （3） the high doses of TSN Ⅱ A and valsartan significantly down-regulated the mRNA expression of AT 1R and protein expression of TGF-beta l and Smad-3 in the hypertrophic myocardium （P〈0.01）, and TGF-betal in valsartan-treated animals was more significantly lower than that in rats treated with TSN Ⅱ A; （4） the two doses of TSN Ⅱ A and valsartan significantly up-regulated the protein expression of Smad-7 in the hypertrophic myocardium （P〈0.01）, and Smad-7 in the animals treated with high-dose TSN Ⅱ A was significantly higher than that in rats treated with valsartan. It is concluded that inhibition of myocardial hypertrophy induced by TSN ⅡA independent of blood pressure. The underlying mechanism might be the down-regulated expression of AT1R mRNA and Smad-3, increased production of Smad-7, and blocking effect of TSN Ⅱ A on TGF betal/Smads signal pathway in local myocardium.     

Biochemical activity and gene analysis of inherited protein C and antithrombin deficiency in two Chinese pedigrees

Background We identified the gene mutations in two Chinese pedigree of type Ⅰ hereditary protein C deficiency and type Ⅰ hereditary antithrombin deficiency.Methods The plasma level of protein C activity (PC: A), protein C antigen ( PC: Ag) , protein S activity, antithrombin activity (AT: A) and antithrombin antigen (AT: Ag) of propositi and two family members were detected using ELISA and chromogenic assay, respectively. All exons and intron-exon boundaries of protein C gene and antithrombin gene were analyzed by direct sequencing of the corresponding amplified PCR products in DNA from the propositus.Results The plasma PC: A and PC: Ag of propositus 1 was 26% and 1.43 mg/dl, respectively. The PC: Ag and PC: A of his father were normal. The decreased PC: A level was seen in his mother and 4 of his maternal pedigree. PS: A and AT: A were all normal in pedigree 1 members. A C5498T heterozygous mutation in exon 3 of protein C gene, resulting in the substitution of Arg for Trp at the 15th amino acid, was identified in propositus 1 and 8 of his relatives. The plasma AT: A and AT: Ag of propositus 2 was 48.6% and 10.4 mg/dl, respectively. The reduced AT: A and AT: Ag levels were found in his father and 5 of paternal pedigree. PC: A, PC: Ag and PS: A were all in normal range. A heterozygous 13387-9G deletion in exon 6 of antithrombin gene was identified in propositus 2. This mutation introduced a frameshift and a premature stop at codon 426 and existed in 6 members of pedigree 2.Conclusion The C5498T heterozygous mutation in exon 3 of protein C gene, first reported in China,leads to type Ⅰ hereditary protein C deficiency. The 13387-9G deletion, a novel mutation, can cause antithrombin deficiency and thrombosis.     

Effects of Kaixin Powder(开心散)on Melatonin Receptor Expression and ~(125)I-Mel Binding Affinity in A Rat Model of Depression

Objective:To explore the effects of Kaixin Powder(开心散,KXP) on melatonin receptor(MR)expression and ~(125)I-Mel binding affinity in a depression rat model.Methods:Seventy-two male Wistar rats were divided into six groups:a blank control group,model group,ramelteon group,KXP high-dosage group(HKXP),medium-dosage group(MKXP) and low-dosage group(LKXP).To establish the depression model,all groups except the blank control group were singly housed and exposed to chronic unpredictable mild stress.Weight gain,sucrose consumption and the open-field test were used to evaluate induction of depression.KXP at 260,130 and 65 mg/(kg·d) was also respectively administered to the rats in the HKXP,MKXP and LKXP groups for 21 days.Ramelteon[0.83 mg/(kg·d)]was given to the positive drug control group.An equivalent volume of physiological saline was given to the blank and model groups.The liquid chip method was used to measure the concentration of plasma melatonin(MT).Mel1a(MT1) and Mel1b(MT2) expression levels were determined by Western blotting.In addition,a radioactive ligand-binding assay was used to analyze the specific binding properties and dynamic characteristics between MR and ~(125)I-Mel.Results:The results of weight gain,sucrose consumption and the open-field test showed that our model successfully produced depressive symptoms and depressive-like behavior.The concentration of plasma MT in the model group decreased significantly at night but increased in the MKXP group(P0.05).The HKXP group showed significantly increased expression of MT1(P0.05);however,the expression of MT2 in all groups exhibited no significant differences(P0.05).The maximum binding capacity(B_(max)) for specific binding between MR and ~(125)I-Mel in the MKXP group was significantly higher than that in the model group(P0.05),but no significant differences were found in the equilibrium dissociation constant(K_d) of each group(P0.05).Conclusions:KXP may have a similar effect as ramelteon.KXP improved depressive-like behavior by increasing the concentration of plasma MT and MT1 expression,thereby increasing three B_(max) of MR to achieve the desired antidepressant effect.     

金属硫蛋白及其基因在人胰腺癌细胞株中的表达及意义

目的比较金属硫蛋白(MT)及其基因在人胰腺癌细胞株和耐药细胞株中的表达,探讨MT表达与胰腺癌细胞耐药的关系.方法采用逆转录聚合酶链反应(RT-PCR)检测MT基因的mRNA表达情况,采用镉/血红蛋白饱和-电化学分析法检测MT表达情况.结果人胰腺癌细胞株中的MT可由MT-1A、MT-1B、MT-1E、MT-1F、MT-1G、MT-1X及MT-2A基因编码:MT-1B、MT-1E、MT-1X及MT-2A基因和MT在人胰腺癌耐药细胞株中的表达水平显著增高(P＜0.05).结论人胰腺癌细胞株中的MT及其基因表达与胰腺癌细胞株的增殖及化疗耐药性相关.     

人MT2A基因在哺乳动物细胞株中的定位与表达

目的 用基因克隆方法构建带FLAG-tag的人金属硫蛋白2A(MT2A)真核表达载体,用其转染HEK 293T,COS-7细胞观察MT2A在增殖细胞内的表达和定位.方法 设计带FLAG-tag的引物,以人MT2A全长cDNA序列的质粒为模板,PCR法扩增MT2A全长序列,然后插入pCDNA 3.1中构建pCDNA 3.1-MT2A-FLAG质粒;将构建的质粒转染至HEK 293T细胞,提取总蛋白,进行Western blot检测;脂质体法转染至COS-7细胞,荧光显微镜下观察MT2A表达的蛋白在细胞内定位.结果 正确构建了pCDNA 3.1-MT2A-FLAG质粒;其在HEK 293T细胞中能有效地表达;免疫荧光实验表明在COS-7细胞中,MT2A主要分布在细胞质内.结论 该研究结果为了解MT2A 在细胞内与其他蛋白质相互作用及功能提供了一定的基础.     

Mitogen-activated protein kinase-dependent apoptosis in norcan-tharidin-treated A375-S2 cells is proceeded by the activation of protein kinase C

Background We have reported that norcantharidin (NCTD) induces human melanoma A375-S2 cell apoptosis and that the activation of caspase and the mitochondrial pathway are involved in the apoptotic process. This study aimed at investigating the roles of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) in A375-S2 cell apoptosis induced by NCTD.Methods We assessed the effects of NCTD on cell growth inhibition using the 3-(4,5-dimethyhhiazol-2-yl)-2,5-dipheyhetrazolium bromide (MTT) assay, DNA fragmentation (DNA agarose gel electrophoresis), and MAPK protein levels (Western blot analysis) in A375-S2 cells. Photomicroscopic data were also collected.Results The NCTD inhibitory effect on A375-S2 cells was partially reversed by MAPK and PKC inhibitors.The expression of phosphorylated JNK and p38 also increased after the treatment with NCTD, and inhibitors of c-Jun NH2 -terminal kinase (JNK) and p38 (SP600125 and SB203580, respectively) had significant inhibitory effects on the upregulation of phosphorylated JNK and p38 expression. Simultaneously, the PKC inhibitor staurosporine blocked the upregulation of phosphorylated JNK and phosphorylated p38, but had little effect on extracellular signal-regulated kinase (ERK) expression.Conclusion These results suggest that the activation of JNK and p38 MAPK promotes the process of NCTDinduced A375-S2 cell apoptosis and that PKC plays an important regulation role in the activation of MAPKs.     

Expression and significance of P53 protein and MDM-2 protein in human gliomas

Background  P53 is one of the most studied tumor suppressors in the cancer research, and over 50% of human tumors carry P53 mutations. MDM-2 is amplified and/or overexpressed in a variety of human tumors of diverse tissue origin. The aim of this study was to examine the expression of P53 protein and MDM-2 protein in gliomas, and to investigate the relationship between the expression of the two proteins and the histopathological grades of glioma. The relationship between MDM-2 protein expression and P53 protein expression was also analyzed.

Methods  The expression of P53 protein and MDM-2 protein was immunohistochemically detected using monoclonal antibodies in 242 paraffin embedded tissues, including 30 normal brain tissues from patients with craniocerebral injury and 212 tissues from patients with primary glioma (grade I–II group: 5 cases of grade I, 119 cases of grade II; and grade III–IV group: 53 cases of grade III, and 35 cases of grade IV).

Results  The P53 positive rate was significantly higher in the glioma groups than in the control group (P <0.0001). The P53 positive rate was significantly higher in glioma tissues of grade III–IV than in glioma tissues of grade I–II group (P=0.001). The MDM-2 positive rate was significantly higher in glioma groups than in the control group (P <0.0001). There was no significant difference in the MDM-2 positive rate between the two glioma groups (P=0.936). The expression of P53 protein was not related to expression of MDM-2 protein (P=0.069)

Conclusions  Overexpression of P53 protein might be related to the occurrence and progression of glioma. Overexpression of MDM-2 protein may play an important role in glioma tumorigenesis, but may not be involved in glioma progression. The overexpression of MDM-2 protein was an early event in malignant transformation of glioma. MDM-2 may be a key player in glioma in its own right.

     

MT2A基因多态性与2型糖尿病的关系

目的:探讨16号染色体长臂（16q）金属硫蛋白(metallothionein,MT)基因多态性与中国北方汉族人群2型糖尿病的关系。方法:采用PCR技术和限制性片段长度多态性(RFLP)的方法检测129名糖尿病患者(病例组)和115名正常对照者（对照组）的MT基因家族中的MT2A基因rs10636位点单核苷酸多态性。结果:rs10636 3种基因型GG、GC和CC的频数分布符合Hardy-Weinberg平衡;MT2A的3种基因型GG、GC和CC的频数分布在病例组和对照组中无统计学意义(χ²=0.202,P>0.05),等位基因G和C的频数分布在病例组和对照组中也无统计学意义(χ²=0.136,P>0.05)。结论:MT2A基因rs10636位点可能与2型糖尿病无关。     

兔脑出血灶周围脑组织MT的表达

Intracerebral hemorrhage frequently occurs in nervous system disease and leads to secondary cerebral lesion. Recent studies have confirmed that metalloth ionein (MT) is involved in the pathogenesis of cerebral hemorrhage. Using immun ohistochemical technique,the author detected the expression of MT in the perih ematomal brain regions in order to research the pathology mechanism of intracer ebral hemorrhage.     

Expression and self-assembly of HCV structural proteins into virus-like particles and their immunogenicity

Background The synthesis of virus-like particles (VLPs) provides an important tool to determine the structural requirements for viral particle assembly and virus-host interactions. Our purpose was to express simultaneously all three structural proteins of hepatitis C virus (HCV) in insect cells to investigate the proteins assembly into VLPs and the imrnunogenicity of these particles. Methods HCV gene sequences encoding the structural proteins C, E1, and E2 were amplified with PCR, and recombinant baculoviruses were constructed using recombinant DNA techniques. The expression of HCV structural proteins in insect cells was analyzed by immunofluoresceoce and SDS-PAGE. The interaction of expressed structural proteins was investigated by immunoprecipitation and immunoblotting. The VLPs in the insect cells were visualized by electron microscopy (EM). VLPs were then purified by sucrose gradient centrifugation and used to immunize BALB/c mice. Antibodies against HCV were tested for in mouse serum samples by an ELISA assay. Results The recombinant baculoviruses reBV/C and reBV/E1-E2 were constructed successfully. Insect cells co-infected with reBV/C and reBV/E1 -E2 expressed HCV C, E1, and E2 proteins with the expected molecular weights of 20kD, 35kD, and 66kD, respectively. The results of immunoprecipitation and immunoblotting assays revealed the coimmunoprecipitation of C, E1, and E2 proteins, indicating association of the three structural proteins. Electron microscopy of insect cells coinfected with reBV/C and reBV/E1-E2 demonstrated spherical particles (40 to 60 nm in diameter) similar to the HCV virions from serum samples or hepatic tissue samples of HCV infected humans. The VLPs were partially purified. Antibodies to HCV were detectable in the serum of mice immunizedwith VLPs. Conclusion HCV structural proteins simultaneously expressed in insect cells can interact with each other and assemble into HCV-like particles, which are shown to be immunogenic in mice.