金属诱导条件下刺猬各组织器官金属硫蛋白含量的比较分析

本文采用Ａｇ－Ｈｂ（血红蛋白）饱和法,Ｃｄ－Ｈｂ饱和法、Ｃｕ－Ｈｂ饱和法及酶联吸附免疫法定量分析刺猬各组织器官中金属硫蛋白含量。结果表明,肌肉注射三种金属盐均有明显的诱导金属硫蛋白合成的作用,诱导强度为Ｃｄ＾２＋＞Ｚｎ＾２＋＞Ｃｕ＾２＋,诱导后金属硫蛋白含量分布规律一致,即肝＞肾＞睾丸＞脾＞心、肺、胃、膀胱、胆汁、大肠、小肠、肌肉、血液,其中镉诱导肝的金属硫蛋白含量每克湿组织约１．８４ｍｇ,为肾的     

亚香棒虫草菌丝体金属硫蛋白的提纯及性质

【目的】探讨锌离子诱导亚香棒虫草(Cordyceps hawkesii)菌丝体金属硫蛋白的产生及性质。【方法】亚香棒虫草菌丝体以18 g/L Zn2+在10 L发酵罐中诱导培养64 h后收集菌丝体,产率为每升发酵液收集12.2 g菌丝体(干重),细胞破碎取上清液通过2次凝胶柱层析,冷冻干燥得到亚香棒虫草菌丝体金属硫蛋白纯品。利用考马斯亮蓝法(Bradford法)进行含量测定,用银饱和分析法结合原子吸收光谱(AAS)测定MT含量,用Ellman’s方法和火焰原子吸收法分别测得巯基含量和结合锌原子数,用电喷雾质谱仪测得分子量,全自动氨基酸分析仪测氨基酸组成。通过对羟基自由基、DPPH自由基和超氧阴离子自由基的清除率试验探讨亚香棒虫草金属硫蛋白的抗氧化活性。【结果】发酵终点金属硫蛋白产量为15.3 mg/g菌丝体湿重。金属硫蛋白的分子量为7 680 Da,每分子蛋白质含有18个巯基、结合4个Zn原子。氨基酸组成分析结果显示,每分子蛋白质共含60个氨基酸,其中含有15个半胱氨酸,且含有组氨酸和芳香族氨基酸。亚香棒虫草金属硫蛋白的抗氧化活性稍强于谷胱甘肽,弱于动物金属硫蛋白。【结论】亚香棒虫草在Zn2+胁迫下能够大量合成金属硫蛋白,且其金属硫蛋白的性质与哺乳动物金属硫蛋白有相似性。     

金属硫蛋白的结构模型

金属硫蛋白是一种低分子量(6,000—10,000),富含半胱氨酸(25—35％),并能结合重金属如Zn、Cd、Cu、Hg或Ag的蛋白质。因其含硫及金属故称为金属硫蛋白(Metallothionein,简称MT)。自1957年Margashes和Vallee在马的肾脏皮质中第一次分离出含有镉的蛋白质以来,在哺乳动物(包括人)、微生物、原核生物蓝绿藻及植物中已先后分离出金属硫蛋白。它是一种诱导性的蛋白质。在实验的动物和培养的细胞中(如Hela细胞、人的上皮细胞等)金属硫蛋白不仅能被重金属离子而且也能为皮质激素诱导而合成。并已证明二者诱导MT的合成是受转录水平调节。     

水生无脊椎动物金属硫蛋白研究进展

金属硫蛋白在水生无脊椎动物中分布广泛、容易被诱导,在水环境生态响应研究中具有重要意义。对水生无脊椎动物金属硫蛋白分类和特性、MT的诱导及影响因素、基因克隆与表达等方面取得的进展进行概述;并对其金属离子调节功能及其在水环境重金属污染监测、重金属污染生物治理和水产养殖等方面的应用潜力进行展望,提出研究中的不足和今后的研究方向。     

金属硫蛋白与单抗SZ51重组蛋白在大肠杆菌菌株BL21(DE3)pLysS中表达的研究

从基因工程水平构建了小鼠金属硫蛋白与抗人活化血小板单抗ＳＺ５１单链抗体的重组基因产物,并在大肠杆菌菌株ＢＬ２１（ＤＥ３）ｐＬｙｓＳ中成功地进行了表达．该重组蛋白以包涵体形式存在于菌体蛋白中,分子量为３８ｋＤ,ＥＬＩＳＡ实验证明该重组蛋白既具有血栓部位活化血小板的单抗活性,又具有小鼠ＭＴ单抗的活性     

镉诱导威廉环毛蚓（Pheretima guillelmi）金属硫蛋白的分离纯化…

蚯蚓威廉环毛（Ｐｈｅｒｅｔｉｍａ ｇｕｉｌｌｅｌｍｉ）经皮下注射ＣｄＣｌ２溶液诱导后,整体匀浆,再经热沉淀、乙醇沉淀后,经凝胶过滤ＳｅｐｈａｄｅｘＧ－５０柱层析,得两个镉结合蛋白峰,分子量依次为４３ｋＤ及１９ｋＤ。这两个组份再分别经ＤＥＡＥＳｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ柱层析,各得三个镉结合蛋白峰。根据光谱学特征、疏基含量及氨基酸组成等分析,表明凝胶过滤第二峰为金属硫蛋白（ＭＴ）,其经ＤＥ     

蛹虫草金属硫蛋白分离纯化及性质研究

研究探讨锌离子胁迫下蛹虫草Cordyceps militaris金属硫蛋白的产生及性质。蛹虫草菌丝体以15g/L Zn2+在10L发酵罐中诱导培养56h后收集,产率为每升发酵液收集12.021g菌丝体（干重）,细胞破碎取上清液通过两次凝胶柱层析,冷冻干燥得到蛹虫草金属硫蛋白纯品。利用Bradford法进行蛋白质含量测定,用银饱和分析法结合原子吸收光谱（AAS）测定MT含量,发酵终点处金属硫蛋白含量为12.876mg/g菌丝体（湿重）。用电喷雾质谱法测得金属硫蛋白的分子量为7 390Da,用Ellman’s方法和火焰原子吸收法分别测得每分子蛋白质含有14个巯基、结合5个Zn原子。氨基酸组成分析结果显示,每分子蛋白质共含57个氨基酸,其中含有13个半胱氨酸,疏水氨基酸占29.8%,且含有组氨酸。以上表明,研究中的蛹虫草金属硫蛋白与哺乳动物金属硫蛋白结构差异较大,但与酵母菌金属硫蛋白结构组成类似。     

二价铅离子与金属硫蛋白相互作用的研究

通过紫外吸收光谱和平衡透析法研究了二价铅离子同脱金属硫蛋白（ａｐｏ－ＭＴ）、锌－金属硫蛋白（Ｚｎ－ＭＴ）的相互作用,证实Ｐｂ（Ⅱ）是以金属巯基复合物（金属巯基比为１∶２）的形式同金属硫蛋白结合,表观离解常数（ＫＤ）为８．７１×１０－７ｍｏｌ／Ｌ．在自由铅浓度达到６．５２×１０－６ｍｏｌ／Ｌ的条件下,铅离子即可将Ｚｎ－ＭＴ上的Ｚｎ完全取代下来．通过ＥＤＴＡ、ＤＴＮＢ竞争反应、圆二色性（ＣＤ）光谱分析,认为Ｐｂ－ＭＴ的金属巯基复合物不同于Ｚｎ－ＭＴ中Ｚｎ与巯基形成的紧密的正四面体结构,而是可能形成一种三级结构相对松散、热力学上不稳定的Ｃｙｓ－Ｓ－Ｐｂ－Ｓ－Ｃｙｓ平面形结构．研究认为金属硫蛋白的两种亚型ＭＴ－Ⅰ、ＭＴ－Ⅱ与Ｐｂ（Ⅱ）的结合能力并无显著差异     

金属硫蛋白的表达调控及其与锌的关系

金属硫蛋白是普遍存在于人和动物组织中的一种金属结合蛋白,它属于机体抗氧化系统的一部分．在氧化应激导致的细胞病理改变中具有重要的保护作用。金属硫蛋白可由多种因子诱导产生,而且它与金属锌密切相关,二者之间的平衡对维持机体正常的生理功能起着重要的作用。     

金属硫蛋白融合蛋白的分离纯化

采用IPTG诱导新1#菌BL21（DE3）（pET21a-MT)高效表达Balb/C小鼠金属硫蛋白融合蛋白,并采用包涵体分离纯化方法及Sephadex G-75柱层析得到SDS－PAGE电泳为一条带的金属硫蛋白融合蛋白收集液。     

TP53, BCL-2, p21Waf1/Cip1 and metallothionein as markers of differentiation, response to treatment and prognosis in neuroblastic tumors

OBJECTIVE: To identify markers of response to therapy in neuroblastic tumors. STUDY DESIGN: A total of 58 patients with neuroblastic tumor (38 neuroblastomas, 13 ganglioneuroblastomas and 7 ganglioneuromas) were included in the study. TP53, BCL-2, p21Waf1/Cip1 and metallothionein were included as a biologic approach to tumor differentiation, response to therapy and prognosis. RESULTS: Patients who died of disease had the following immunophenotype: BCL-2 (9 of 10), nuclear TP53 (7 of 10) and metallothionein (7 of 10). TP-53 expression was related to clinical stage (p = 0.062) and disease outcome (p = 0.0218). All patients in whom treatment failed expressed metallothionein (3 of 3). CONCLUSION: TP53, BCL-2, p21Waf1/Cip1 and metallothionein had limited value reflecting tumor maturation (differentiation) or predicting response to therapy. Only nuclear TP53 accumulation may be relevant in patient's prognosis.     

Serum metallothionein in newly diagnosed patients with childhood solid tumours

Tumour markers are substances produced by malignant cells or by the organism as a response to cancer development. Determination of their levels can, therefore, be used to monitor the risk, presence and prognosis of a cancer disease or to monitor the therapeutic response or early detection of residual disease. Time-consuming imaging methods, examination of cerebrospinal fluid or tumour tissue and assays for hormones and tumour markers have been used for cancer diagnosis. However, no specific marker for diagnosis of childhood solid tumours has been discovered yet. In this study, metallothionein (MT) was evaluated as a prospective marker for such diseases. Serum metallothionein levels of patients with childhood solid tumours were determined using differential pulse voltammetry - Brdicka reaction. A more than 5-fold increase in the amount of metallothionein was found in sera of patients suffering from cancer disease, compared with those in sera of healthy donors. The average metallothionein level in the sera of healthy volunteers was 0.5 ± 0.2 μmol ? dm?3 and was significantly different (p<0.05, determined using the Schefe test) from the average MT level found in serum samples of patients suffering from childhood solid tumours (3.4 ± 0.8 μmol ? dm?3). Results found in this work indicate that the MT level in blood serum can be considered as a promising marker for diagnostics, prognosis and estimation of therapy efficiency of childhood tumours.     

Hepatic metallothionein synthesis in neonatal mottled-brindled mutant mice

Mottled-brindled mutant mice did not display the elevated hepatic metallothionein synthesis normally observed in 2- to 6-day-old wild-type mice. This difference between normal and mutant mice was not due to a decreased ability to synthesize metallothionein in the liver, since hepatic metallothionein synthesis was inducible in response to copper, cadmium, zinc, or hydrocortisone administration to neonatal mutant mice. Hydrocortisone treatment resulted in increased metallothionein synthesis in liver of mutant mice but had no ameliorative effect on the mottled-brindled disease.This work was supported by Contract DE AM03 76 SF00012 between the Department of Energy and the Regents of the University of California. JEP is the recipient of a National Research Service Award from the National Institutes of Health.     

Evaluation of the clastogenicity and anticlastogenicity of the carotenoid bixin in human lymphocyte cultures

Carotenoids are regarded as effective antioxidants, antimutagenic and anticarcinogenic agents. Annatto, a red-yellow extract obtained from seeds of Bixa orellana L. is a mixture of several carotenoids and one of them bixin (BXN), is known as its major coloring compound. Studies on BXN clastogenicity and anticlastogenicity in cultured human lymphocytes have not been reported so far. Therefore, the present study was undertaken to investigate the ability of BXN to induce chromosomal aberrations in human lymphocytes in vitro and to examine the possible anticlastogenic effect of this carotenoid in chromosomal damage induced by the clastogen cisplatin (cDDP). Human blood samples were obtained from six healthy, non-smoking volunteers; two females and four males aged 18-35 years. The concentrations of BXN (1.0; 2.5; 5.0 or 10 microg/mL) tested in combination with cDDP were established on the basis of mitotic index (MI) measurements. The data showed that BXN was not cytotoxic or clastogenic, when compared to untreated control. A marked decrease in the MI values compared to the untreated control and an increased percentage of aberrant metaphases was seen in all cultures treated with cDDP. The carotenoid efficiency in reducing the inhibitory effect of cDDP on lymphocyte MI is concentration-dependent. Cultures simultaneously treated with BXN and cDDP showed a statistically significant reduction in total chromosomal aberrations and aberrant metaphases. In our experiments, BXN may have acted as an antioxidant by intercepting free radicals generated by cDDP. The data obtained in the present study suggest that dietary carotenoids may act as protective agents against clastogenic effects of antitumor agents. However, extensive studies are necessary to elucidate the mechanism of action of BXN before its therapeutic use.     

Regulation of caspase activation and cis-diamminedichloroplatinum(II)-induced cell death by protein kinase C

Activation of caspases is critical for the induction of apoptosis. We have shown previously that cell death mediated by the anticancer agent cis-diamminedichloroplatinum(II) (cDDP) is influenced by the protein kinase C (PKC) signal transduction pathway. In the present study, we have examined whether regulation of cDDP sensitivity by PKC involves caspase activation. cDDP caused a time- and concentration-dependent increase in the generation of the catalytic fragment (CF) of novel (n) PKCdelta, nPKCepsilon, and atypical (a) PKCzeta but had little effect on conventional (c) PKCalpha. Cleavage of PKC isozymes was associated with the activation of caspase-3 and -7 but not of caspase-2. PKC activators enhanced cDDP-induced cleavage of these isozymes and activation of caspase-3. Rottlerin, an inhibitor of nPKCdelta, blocked caspase-3 activation and proteolytic cleavage of nPKCdelta by cDDP. Bryostatin 1, which elicits a biphasic concentration-response in potentiating cell death by cDDP, exhibited a similar biphasic effect on cDDP-induced activation of caspase-3 and caspase-7 and the cleavage of poly(ADP-ribose) polymerase; while 1 nM bryostatin 1 induced maximum activation of these caspases, 1 microM bryostatin 1 had little effect. z-DEVD-fmk, an inhibitor of caspase-3-like proteases, prevented cDDP-induced cell death. Bryostatin 1 also induced a similar biphasic down-regulation of nPKCdelta but not of cPKCalpha or nPKCepsilon. These results suggest that nPKCdelta not only acts downstream of caspases but also regulates the activation of caspases and that the biphasic concentration response of bryostatin 1 on cDDP-induced cell death could be explained by its distinct effect on nPKCdelta down-regulation and caspase activation.     

Involvement of protein kinase C-delta in DNA damage-induced apoptosis

We have previously shown that the protein kinase C (PKC) signal transduction pathway regulates cell death by the DNA damaging agent cis-diamminedichloroplatinum(II) (cDDP). In the present study we have investigated how PKC influences the sequence of events that are triggered by cDDP-induced DNA damage. cDDP caused activation of caspases-8, -9, -3, -7 and cleavage of PKCdelta. Rottlerin, a selective inhibitor of novel PKCdelta, blocked activation of caspases, proteolytic activation of PKCdelta and cell death induced by cDDP. In contrast, G? 6976, an inhibitor of conventional PKCalpha and betaI, did not prevent cDDP-induced caspase activation and cDDP cytotoxicity. In HeLa cells, PKCdelta was distributed both in the cytosol and heavy membrane (HM) fraction containing mitochondria. While caspase-8 was primarily cytosolic, a small amount of caspases-9, -7 and -3 could be detected in the HM fraction. cDDP caused a time-dependent increase in Cytochrome c release from the mitochondria and processing of both cytosolic and membrane-associated caspases, as well as proteolytic cleavage of PKCdelta. Rottlerin attenuated late but not early release of Cytochrome c by cDDP. It, however, inhibited activation of caspases and proteolytic cleavage of PKCdelta in both cytosolic and HM fractions. The antiapoptotic effect of rottlerin was evident when it was added together with or following cDDP addition but not when added after cDDP was removed from the medium. Thus, the PKCdelta inhibitor acts at an early stage of the cDDP-induced cell death pathway that precedes caspase activation.     

Identification Keratin 1 as a cDDP-resistant protein in nasopharyngeal carcinoma cell lines

Multidrug resistance (MDR) to anticancer drugs is a major obstacle to successful chemotherapy of tumors. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cis-diamminedichloroplatinum (CNE2/cDDP) were established from human nasopharyngeal carcinoma (NPC) cell lines CNE2. Comparative proteomics involving 2-dimensional gel electrophoresis (2-DE) and ESI-Q-TOF-MS were performed on protein extracted from CNE2 and CNE2/cDDP cell lines to screen drug resistance-related proteins. Keratin 1 (KRT1), cathepsin D (CTSD) and annexin a5 (ANXA5) were identified as three proteins showing higher expression in CNE2/cDDP compared to CNE2. Furthermore, suppression of KRT1 expression by siRNA resulted in decreased MDR in siRNA-CNE2/cDDP cells. And upregulation of KRT1 could result in increased of drug resistance in NPC cell lines. Taken together, KRT1 protein and its activity levels were higher in cDDP-resistant NPC cell lines compared to their parental cell lines. These data clearly linked KRT1 and cDDP resistance mechanisms. KRT1 could serve as a biomarker for chemotherapy sensitivity of NPC.