Elevated Cu/Zn-SOD exacerbates radiation sensitivity and hematopoietic abnormalities of Atm-deficient mice

Patients with the genetic disorder ataxia-telangiectasia (A-T) display a pleiotropic phenotype that includes neurodegeneration, immunodeficiency, cancer predisposition and hypersensitivity to ionizing radiation. The gene responsible is ATM, and ATM:-knockout mice recapitulate most features of A-T. In order to study the involvement of oxidative stress in the A-T phenotype, we examined mice deficient for Atm and overexpressing human Cu/Zn superoxide dismutase (SOD1). We report that elevated levels of SOD1 exacerbate specific features of the murine Atm- deficient phenotype, including abnormalities in hematopoiesis and radiosensitivity. The data are consistent with the possibility that oxidative stress contributes to some of the clinical features associated with the A-T phenotype.     

牛血Cu/Zn-SOD的热稳定性

研究了不同的Cu^2 和Zn^2 浓度配比对SOD热稳定性的影响,并测定了SOD活性及利用考马斯亮蓝D250法测定蛋白质含量。结果表明在Cu^2 、Zn^2 分别为5．4mmol／1．和3．6mmol／L时SOD的活性最高,达到了92．6％;在75℃时大部分杂蛋白变性沉淀,SOD的比活力最高。最后确定在加入5．4mmol／L．Cu^2 ,3．6mmol／LZn^2 条件下,75℃下热变性,可以取得比较高活性和比活性的SOD。从而为寻找更简便、经济的提纯方法提供参考依据。     

羊血铜锌超氧化物歧化酶(Cu-ZnSOD)的分离纯化

目的:对羊血进行提取SOD。方法:采用有机溶剂去除血红蛋白、热变性、丙酮沉淀、DEAE-32离子交换柱层析的方法,对羊红细胞Cu,Zn-SOD进行分离纯化。利用非变性凝胶电泳NBT活性染色鉴定SOD,SDS-PAGE测定分子量。结果:表明1 000ml羊血中得到SOD干粉1 257mg,总活力为79 453U,比活力为3 871.4U/mg。活性染色结果证明羊血SOD有2条带,表明已达到电泳纯。SDS-PAGE测得SOD亚基分子量分别为16.71kDa、15.97kDa。H2O2对羊血CuZn-SOD活性有抑制作用,通过紫外光谱扫描,羊血SOD在231nm处有最大吸收峰。     

Rosiglitazone improves survival and hastens recovery from pancreatic inflammation in obese mice

Obesity increases severity of acute pancreatitis (AP) by unclear mechanisms. We investigated the effect of the PPAR-gamma agonist rosiglitazone (RGZ, 0.01% in the diet) on severity of AP induced by administration of IL-12+ IL-18 in male C57BL6 mice fed a low fat (LFD) or high fat diet (HFD), under the hypothesis that RGZ would reduce disease severity in HFD-fed obese animals. In both LFD and HFD mice without AP, RGZ significantly increased body weight and % fat mass, with significant upregulation of adiponectin and suppression of erythropoiesis. In HFD mice with AP, RGZ significantly increased survival and hastened recovery from pancreatic inflammation, as evaluated by significantly improved pancreatic histology, reduced saponification of visceral adipose tissue and less severe suppression of erythropoiesis at Day 7 post-AP. This was associated with significantly lower circulating and pancreas-associated levels of IL-6, Galectin-3, osteopontin and TIMP-1 in HFD + RGZ mice, particularly at Day 7 post-AP. In LFD mice with AP, RGZ significantly worsened the degree of intrapancreatic acinar and fat necrosis as well as visceral fat saponification, without affecting other parameters of disease severity or inflammation. Induction of AP lead to major suppression of adiponectin levels at Day 7 in both HFD and HFD + RGZ mice. In conclusion, RGZ prevents development of severe AP in obese mice even though it significantly increases adiposity, indicating that obesity can be dissociated from AP severity by improving the metabolic and inflammatory milieu. However, RGZ worsens selective parameters of AP severity in LFD mice.     

Effect of overexpression of human Cu/Zn-SOD on activation-induced lymphocyte proliferation and apoptosis

The process of lymphocyte proliferation and apoptosis is known to be linked to oxidative stress. In the present study, we have used a new transgenic mouse model to investigate the effect of human Cu/Zn superoxide dismutase (Cu/Zn-SOD) overexpression on activation-induced lymphocytes proliferation and apoptosis. Cu/Zn-SOD activity was 3.5-fold higher in the spleen of the transgenic mice overexpressing Cu/Zn-SOD (Tg-Cu/Zn-SOD) compared to the wild-type littermates. Proliferative response of lymphocytes to lipopolysaccharide (LPS), Concanavalin A (Con A), and anti-CD3 was measured by [3H]-thymidine incorporation. Activation-induced apoptosis was determined by incubating the T cells with anti-CD3 (primary stimulus) for 72 h, followed by restimulation with Con A (secondary stimulus) for various times. Apoptosis was assessed by measuring DNA fragmentation using a spectrofluorimetric assay and monitoring the expression of the specific apoptotic markers (Fas/CD95 receptor and Fas/CD95 ligand (Fas-L) using flow cytometry. There was no significant difference in proliferative response of lymphocytes to LPS, Con A, or anti-CD3 in transgenic mice overexpressing human Cu/Zn superoxide dismutase (Tg-Cu/Zn-SOD) compared to wild-type littermates. In addition, no significant difference was observed in lymphocyte populations and subsets between Tg-Cu/Zn-SOD mice and wild-type littermates. However, splenic T cells from Tg-Cu/Zn-SOD mice exhibited a significantly (p <.05) higher level of activation-induced DNA fragmentation than T cells from wild-type littermates. The increase in DNA fragmentation was paralleled with an increase in the proportion of T cells expressing Fas and Fas-L molecules. The possible consequences of Cu/Zn-SOD overproduction on activation-induced apoptosis are discussed.     

Targeted imaging of colonic tumors in smad3-/- mice discriminates cancer and inflammation

The peripheral benzodiazepine receptor (PBR) is a trans-mitochondrial membrane protein that modulates steroid biosynthesis. Recently, up-regulation and nuclear localization of PBR has been shown to be associated with colon, prostate, and breast cancer. PBR has been targeted by the exogenous synthetic ligand, PK11195, for various purposes including imaging. To capitalize on these observations, we developed a high-throughput, noninvasive, in vivo imaging approach to detect spontaneously arising colonic tumors in mice using a novel PBR-targeted molecular imaging agent (NIR-conPK11195). NIR-conPK11195 localized and was retained in colonic adenomas and carcinomas in Smad3(-/-) mice but not in non-neoplastic hamartomas or chronically inflamed colonic tissue. Using a fluorescence signal-to-noise ratio of > or =4-fold 13 h after injection of the agent, we detected colonic tumors with a sensitivity of 67% and a specificity of 86% in a cohort of 37 Smad3(-/-) mice and control littermates. Furthermore, using oral administration of dextran sulfate to induce colonic inflammation, we showed that the clearance profile of NIR-conPK11195 distinguished transient uptake in inflammatory tissue from longer term retention in tumors. Taken together, these results indicate that NIR-conPK11195 is a promising optical molecular imaging tool to rapidly screen for colonic tumors in mice and to discriminate inflammation from cancer.     

酿酒酵母超氧物歧化酶(SOD)基因的克隆和表达

通过PCR扩增技术从酿酒酵母中得到了Cu,zn—SOD的结构基因,此基因被亚克隆到大肠杆菌质粒载体pT7—7．得到重组质粒pT7－7：SOD。利用EcoRI和Pstl酶切pT7-7::SOD质粒．经琼脂糖凝腔电泳,DEAE-滤膜回收Cu。zn—SOD结构基因片段,将其亚克隆到M13中．并转化大肠杆菌,得到了重组质粒M13-::t SOD,酶切和纯化后的SOD基因,定向克隆到酵母质粒载体pHz－8的smal和EcoRI位点上,构建成重组质粒pHZ－8－l。经转化酵母受体菌ZH-l和DP—l后得到了转化子．来自于ZH—l的转化子在非选择性条件下培养40世代后仍有95％以上细胞保留重组质粒。而来自于DP-1的转化子很不稳定。经蛋白提取、聚丙烯酰胺凝胶电泳和酶活性测定结果表明,来自于zH－1转化子中SOD的表达量约为细胞可溶性蛋白的15％．并具有生物活性。     

Cu/Zn-SOD基因植物表达载体的构建及其在烟草中的表达

为研究Cu/Zn-SOD基因在提高转基因植物抗逆性方面的作用,从一株地热芽孢杆菌(Geobacillus)中克隆得到Cu/Zn-SOD基因,以pZP211质粒为表达载体,构建了植物表达载体pZP211-Cu/ZnSOD,并通过农杆菌介导对烟草进行遗传转化.经PCR检测证明已获得转Cu/Zn-SOD基因的烟草.进而测定转基因烟草的SOD活力,结果表明Cu/Zn-SOD基因在烟草中高效表达.对转基因烟草进行耐盐性检测,证明Cu/Zn-SOD基因确实能够提高烟草对盐胁迫的耐受性.     

Chronic inflammation and protection from acute hepatitis in transgenic mice expressing TNF in endothelial cells

Endothelial activation is an important feature of many inflammatory diseases and has been implicated as the cause of vascular complications in disorders such as diabetes, atherosclerosis, and transplant rejection. One of the most potent activators of the endothelium is TNF, which can also be expressed by endothelial cells, causing a permanent, autocrine stimulatory signal. To establish a model of continuous endothelial activation and to elucidate the role of endothelial derived TNF in vivo, we generated transgenic mice expressing a noncleavable transmembrane form of TNF under the control of the endothelial-specific tie2 promoter. Adult tie2-transmembrane TNF-transgenic mice developed chronic inflammatory pathology in kidney and liver, characterized by perivascular infiltration of mononuclear cells into these organs. Along with the infiltrate, an up-regulation of the adhesion molecules ICAM-1 and VCAM-1, but not E-selectin, in the endothelium was observed. Despite predisposition to chronic inflammation these mice were protected from immune-mediated liver injury in a model of Con A-induced acute hepatitis. Although the blood levels of soluble TNF and IFN-gamma were increased in transgenic animals after challenge with Con A, no damage of hepatocytes could be detected, as assessed by the lack of increase in plasma transaminase activities and the absence of TUNEL staining in the liver. We conclude that expression of transmembrane TNF in the endothelium causes continuous endothelial activation, leading to both proinflammatory and protective events.     

Cockroach allergen-induced eosinophilic airway inflammation in HLA-DQ/human CD4(+) transgenic mice

Airway eosinophilic inflammation is a characteristic feature of allergic asthma. Exposure to allergens produced by the German cockroach (Blattella germanica) is a risk factor for allergic disease in genetically predisposed individuals, and has been linked to an increase in asthma morbidity among cockroach-sensitive inner city children. To determine the role and contribution of specific HLA class II in the pathogenesis of allergic airway inflammation in cockroach-induced asthma, we generated double-transgenic, double-knockout mice expressing human HLA-DQ8, HLA-DQ6, and CD4 molecules in the absence of mouse class II and mouse CD4. Mice were actively immunized and later challenged intranasally with cockroach allergen extract. These mice developed bronchoalveolar lavage fluid (BALF) eosinophilia and pulmonary eosinophilia. This was accompanied by an increase in total protein levels, IL-5, and IL-13 in BALF. There were also elevated levels of cockroach-specific serum IgG1 and total serum IgE. Histological analysis revealed peribronchial and perivascular eosinophilic inflammation in cockroach-treated mice. Other pathologic changes in the airways were epithelial cell hypertrophy and mucus production. Treatment with anti-DQ mAb significantly reduced pulmonary and BALF eosinophilia in cockroach allergen-sensitized mice. Abeta(0) mice and transgenic mice expressing human CD4 molecule alone (without class II) or human HLA-DQ8 molecule (without CD4) treated in the same fashion showed no eosinophilia in bronchoalveolar fluid and no pulmonary parenchymal inflammation. Our results provide direct evidence that HLA-DQ molecules and CD4 T cells mediate cockroach-induced eosinophilic inflammation in the airways.     

Intracellular Cu/Zn superoxide dismutase (Cu/Zn-SOD) from hard clam Meretrix meretrix: its cDNA cloning, mRNA expression and enzyme activity

Hard clam (Meretrix meretrix) is an economically important bivalve in China. In the present study, a gene coding for an intracellular Cu/Zn-SOD was cloned and characterized from hard clam. The full-length cDNA of this Cu/Zn-SOD (designated as Mm-icCuZn-SOD) consisted of 1,383?bp, with a 462-bp of open reading frame (ORF) encoding 153 amino acids. Several highly conserved motifs, including the Cu/Zn binding sites [H(46), H(48), H(63), and H(119) for Cu binding; H(63), H(71), H(80), and D(83) for Zn binding], an intracellular disulfide bond and two Cu/Zn-SOD signatures were identified in Mm-icCu/Zn-SOD. The deduced amino acid sequence of Mm-icCu/Zn-SOD has a high degree of homology with the Cu/Zn-dependent SODs from other species, indicating that Mm-icCu/Zn-SOD should be a member of the intracellular Cu/Zn-dependent SOD family. Real-time PCR analysis showed that the highest level of Mm-icCu/Zn-SOD expression was in the hepatopancreas, while the lowest level occurred in the hemocytes. Hard clam challenged with Vibrio anguillarum showed a time-dependent increase in Mm-icCu/Zn-SOD expression that reached a maximum level after 6?h. Mm-icCu/Zn-SOD purified as a recombinant protein expressed in E. coli retained a high level of biological activity, 83?% after 10?min incubation at 10–50?°C, and more than 87?% after incubation in buffers with pH values between 2.2 and 10.2. These results indicated that Mm-icCu/Zn-SOD may play an important role in the innate immune system of hard clam.     

Identification and characterization of an intracellular Cu,Zn-superoxide dismutase (icCu/Zn-SOD) gene from clam Venerupis philippinarum

Superoxide dismutase (SOD, EC 1.15.1.1) represents one kind of enzyme involved in scavenging the high level of reactive oxygen species (ROS) into molecular oxygen and hydrogen peroxide. In the present study, the intracellular Cu/Zn-SOD gene (icCu/Zn-SOD) of Venerupis philippinarum (denoted as VpSOD) was identified from haemocytes by homology cloning and RACR approaches. The full-length cDNA of VpSOD consisted of 910 nucleotides with a canonical polyadenylation signal sequence AATAAA, a polyA tail, and an open-reading frame of 465 bp encoding 154 amino acids. The deduced amino acid of VpSOD shared high similarity with the icCu/Zn-SODs from other species, indicating that VpSOD should be a new member of icCu/Zn-SOD family. Several highly conserved motifs including Cu, Zn binding sites (H⁴⁶, H⁴⁸, H⁶³, H¹²⁰ for Cu binding, and H⁶³, H⁷¹, H⁸⁰, D⁸³ for Zn binding), intracellular disulfide bond and two Cu, Zn SOD signatures were also identified in VpSOD. The temporal expression of VpSOD in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of VpSOD mRNA was up-regulated rapidly at 6 h post-infection and reached 18-fold of the control group. After a drastic decrease at 12 h, the expression level increased again and reached 22-fold to that in the control group at 96 h post-infection. All these results indicated that VpSOD was an acute-phase protein involved in the immune responses of V. philippinarum.     

Cellular response of antioxidant metalloproteins in Cu/Zn SOD transgenic mice exposed to hyperoxia

Ceruloplasmin, metallothionein, and ferritin are metal-binding proteins with potential antioxidant activity. Despite evidence that they are upregulated in pulmonary tissue after oxidative stress, little is known regarding their influence on trace metal homeostasis. In this study, we have used copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) transgenic-overexpressing and gene knockout mice and hyperoxia to investigate the effects of chronic and acute oxidative stress on the expression of these metalloproteins and to identify their influence on copper, zinc, and iron homeostasis. We found that the oxidative stress-mediated induction of ceruloplasmin and metallothionein in the lung had no effect on tissue levels of copper, iron, or zinc. However, Cu/Zn SOD expression had a marked influence on hepatic copper and iron as well as circulating copper homeostasis. These results suggest that ceruloplasmin and metallothionein may function as antioxidants independent of their role in trace metal homeostasis and that Cu/Zn SOD functions in copper homeostasis via mechanisms distinct from its superoxide scavenging properties.     

Blood glucose level and survival in streptozotocin-treated human chymase transgenic mice

A growing body of evidence suggests the potential role of chymase in organ injury in diabetes. We investigated blood glucose levels and survival in transgenic mice carrying the human chymase gene (Tg). Intraperitoneal injections of streptozotocin (STZ) (200, 100, 75 and 50 mg/kg in total, i.p.) were given to uninephrectomized Tg mice and wild-type C57BL/6 (BL) mice. Before STZ injection, the Tg mice had significantly lower body weights and slightly higher systolic blood pressure as compared with the BL mice. STZ-treated Tg mice showed significantly higher postprandial blood glucose levels as compared with the STZ-treated BL mice. The survival prevalence of STZ-treated Tg mice was zero, whereas BL mice showed a value of 40% until 42 days. STZ (100, 75 or 50 mg/kg, i.p.)-treated Tg mice also showed a similar pattern as compared with the STZ-treated BL mice. These data suggest that human chymase contributes to blood glucose levels and mortality during the progression of diabetes.     

Diminished serotonin uptake in platelets of transgenic mice with increased Cu/Zn-superoxide dismutase activity.

Reduced levels of the neurotransmitter serotonin in blood platelets is a clinical symptom characteristic of individuals with Down's syndrome. To investigate the possible involvement of the Cu/Zn-superoxide dismutase (CuZnSOD) gene, which resides at the Down locus on chromosome no. 21, in the etiology of that symptom, we examined blood platelets of transgenic mice harboring the human CuZnSOD gene. It was found that platelets of transgenic CuZnSOD animals, which overexpress the transgene, contain lower levels of serotonin than nontransgenic littermate mice, due to a reduced rate of uptake of the neurotransmitter by the dense granules of the platelets. We found that the pH gradient (delta pH) across the dense granule membrane, which is the main driving force for serotonin transport, was diminished in dense granules of transgenic-CuZnSOD. Furthermore, a significantly lower than normal serotonin accumulation rate was also detected in dense granules isolated from blood platelets of Down's syndrome individuals. These findings suggest that CuZnSOD gene dosage is affecting the dense granule transport system and is thereby involved in the depressed level of blood serotonin found in patients born with Down's syndrome.     

Aberrant neuronal and mitochondrial proteins in hippocampus of transgenic mice overexpressing human Cu/Zn superoxide dismutase 1

Mutations of Cu/Zn superoxide dismutase 1 (SOD1), a metalloenzyme catalyzing the conversion of superoxide anion to hydrogen peroxide (H(2)O(2)), are linked to motor neuron degeneration. Transgenic mouse strains overexpressing wild-type human SOD1 (Tg-SOD1) were shown to have mitochondrial swelling, vacuolization, or learning and memory deficits and are widely used for biochemical, genetic, and cognitive studies; this, along with the advent of advanced proteomic methods, made us investigate protein expression in hippocampus. Hippocampal tissues of wild-type, hemizygous, and homozygous Tg-SOD1 mice were isolated and used for two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight identification. We identified several synaptosomal, neuronal, antioxidant, and mitochondrial proteins in hippocampus, and expression levels of syntaxin-binding protein 1, N-ethylmaleimide-sensitive factor, synaptosomal-associated protein 25, dynamin-1, neurofilament triplet L protein, neurofilament triplet M protein, neuronal tropomodulin, and neuronal protein 25 were significantly decreased in Tg-SOD1. None of the antioxidant proteins were altered except mouse SOD1. Mitochondrial ATP synthase alpha/beta chain and elongation factor Tu were aberrant in Tg-SOD1. We conclude that derangement of neuronal and mitochondrial proteins may indicate synaptosomal and neuronal loss in Tg-SOD1 hippocampus, already reported in morphological terms. This observation is of relevance to understanding brain deficits in Down syndrome, as SOD1 is encoded on chromosome 21.     

Altered expression of hypothetical proteins in hippocampus of transgenic mice overexpressing human Cu/Zn-superoxide dismutase 1

Background  

The rapid completion of genome sequences has created an infrastructure of biological information and provided essential information to link genes to gene products, proteins, the building blocks for cellular functions. In addition, genome/cDNA sequences make it possible to predict proteins for which there is no experimental evidence. Clues for function of hypothetical proteins are provided by sequence similarity with proteins of known function in model organisms.     

Altered expression of hypothetical proteins in hippocampus of transgenic mice overexpressing human Cu/Zn-superoxide dismutase 1

Background  

Cu/Zn-superoxide dismutase 1 (SOD1), encoded on chromosome 21, is a key enzyme in the metabolism of reactive oxygen species (ROS) and pathogenetically relevant for several disease states including Down syndrome (DS; trisomy 21). Systematically studying protein expression in human brain and animal models of DS we decided to carry out "protein hunting" for hypothetical proteins, i.e. proteins that have been predicted based upon nucleic sequences only, in a transgenic mouse model overexpressing human SOD1.