Adenovirus-mediated transfer of the 1-cys peroxiredoxin gene to mouse lung protects against hyperoxic injury

1-Cys peroxiredoxin (1-cysPrx) is a novel antioxidant enzyme that has been shown to reduce a broad spectrum of peroxides including phospholipid hydroperoxides. We tested the hypothesis that adenovirus-mediated transfer of the 1-cysPrx gene can protect lungs of mice from oxidant injury. Mice infected with AdLacZ/AdNull were used as a control (AdCon). X-galactosidase staining revealed widespread expression of the LacZ gene in airways and lung alveoli. Compared with AdCon, 1-cysPrx expression was increased about twofold at 3 days after adenovirus infection. Mice with increased Prx expression showed less loss of body weight and longer survival during exposure to 100% O(2) or to 85% O(2) for 4 days followed by 100% O(2). At 72 h of 100% O(2) exposure, AdPrx infection protected mouse lungs from injury as indicated by less pleural effusion, lower lung wet/dry weight, less protein and fewer nucleated cells in bronchoalveolar lavage fluid, and lower content of thiobarbituric acid-reactive substances and protein carbonyls in lung homogenate. These findings show that increased expression of 1-cysPrx through adenovirus-mediated gene transfer protects mouse lungs from hyperoxic injury and delays death.     

1-Cys peroxiredoxin knock-out mice express mRNA but not protein for a highly related intronless gene

1-Cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin family with a single conserved cysteine, is a unique antioxidant enzyme. We have generated mice in which the 1-cysPrx gene has been inactivated; they are viable and fertile. Mice have a highly related intronless gene (1-cysPrx-P1, GenBank accession number AF085220) with the same length of open reading frame (224 aa) as 1-cysPrx but located on a different chromosome. Since the product of this gene possibly could mimic 1-cysPrx function, we compared expression of 1-cysPrx and 1-cysPrx-P1 in mouse tissues by real-time polymerase chain reaction and Western blot. 1-cysPrx mRNA and protein were expressed in all mouse tissues that were examined with the highest expression level in lung. 1-cysPrx-P1 mRNA was expressed only in testis. In the 1-cysPrx 'knock-out' mouse, 1-cysPrx-P1 mRNA expression level was similar to the wild type but protein expression was not detected. Thus, mouse 1-cysPrx-P1 is an mRNA-expressed pseudogene that does not result in detectable protein in vivo.     

Peroxiredoxin 6, a 1-Cys peroxiredoxin, functions in antioxidant defense and lung phospholipid metabolism

Peroxiredoxin 6 (Prdx6), a bifunctional 25-kDa protein with both GSH peroxidase and phospholipase A2 activities, is the only mammalian 1-Cys member of the peroxiredoxin superfamily and is expressed in all major organs, with a particularly high level in lung. Prdx6 uses GSH as an electron donor to reduce H2O2 and other hydroperoxides including phospholipid hydroperoxides at approximately 5 micromol/mg protein/min with K1 approximately 3 x 10(6) M(-1) s(-1). Oxidation of the Cys47 to a sulfenic acid during catalysis requires piGST-catalyzed glutathionylation and reduction with GSH to complete the enzymatic cycle. Prdx6 stably overexpressed in cells protected against oxidative stress, whereas antisense treatment resulted in oxidant stress and apoptosis. Adenoviral-mediated overexpression of Prdx6 in mouse lungs protected against the toxicity of hyperoxia, whereas Prdx6-null mice were more sensitive to the effects of hyperoxia or paraquat. We postulate that Prdx6 functions in antioxidant defense mainly by facilitating repair of damaged cell membranes via reduction of peroxidized phospholipids. The PLA2 activity of Prdx6 is Ca2+ independent and maximal at acidic pH. Inhibition of PLA2 activity results in alterations of lung surfactant phospholipid synthesis and turnover. Thus, Prdx6, a unique mammalian peroxiredoxin, is an important antioxidant enzyme and has a major role in lung phospholipid metabolism.     

Antisense targeting of CXXC finger protein 1 inhibits genomic cytosine methylation and primitive hematopoiesis in zebrafish

CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is a component of the Set1 histone methyltransferase complex. Mice lacking CFP1 suffer a peri-implantation lethal phenotype, and CFP1-deficient embryonic stem cells are viable but unable to differentiate and exhibit a 60-80% decrease in genomic cytosine methylation. A zebrafish homolog of CFP1 has been identified, is approximately 70% similar to murine CFP1, and is widely expressed during development. Zebrafish embryos treated with a zCFP1 antisense morpholino oligonucleotide had little or no circulating red blood cells and exhibited abnormal yolk sac morphology at 48 h post-fertilization. Many of the antisense-treated zebrafish also exhibited cardiac edema, and 14% were dead at 24 h post-fertilization. Morphant zebrafish also exhibited elevated levels of apoptosis, particularly in the intermediate cell mass, the site of primitive erythropoiesis, as well as aberrations in vascular development. Genomic DNA isolated from morphant embryos exhibited a 60% reduction of global genomic cytosine methylation. A similar phenotype was observed with an independent zCFP1 antisense morpholino oligonucleotide, but not following injection of an unrelated control oligonucleotide. The morphant phenotype was rescued when mRNA encoding murine CFP1 was co-injected with the antisense oligonucleotide. Genomic data base analysis reveals the presence of a second version of zebrafish CFP1 (zCFP1b). However, the morphant phenotype observed following specific depletion of zCFP1 indicates that these related genes have nonredundant functions controlling normal zebrafish hematopoiesis and epigenetic regulation. These findings establish the importance of CFP1 during postgastrulation development.     

L-PhGPx expression can be suppressed by antisense oligodeoxynucleotides

Phospholipid hydroperoxide glutathione peroxidase (PhGPx) directly reduces hydroperoxides of phospholipid and cholesterol to their corresponding alcohols. There are two forms of PhGPx: L-PhGPx localizes in mitochondria and S-PhGPx in cytosol. Antisense oligodeoxynucleotides can inhibit specific protein expression. We tested the hypothesis that antisense oligodeoxynucleotides could be designed to inhibit PhGPx expression and thereby sensitize cells to lipid peroxidation induced by singlet oxygen. We chose P4 cells, a cell line established from L-PhGPx cDNA transfected MCF-7 cells, as our cell model. Lipid peroxidation was induced by singlet oxygen generated by Photofrin and visible light. We found that the antisense oligodeoxynucleotide (5' GCCGAGGCTCATCGCGGCGG 3') was effective in suppressing L-PhGPx mRNA, PhGPx protein, and activity. This antisense oligodeoxynucleotide did not interfere with S-PhGPx. When cells were exposed to singlet oxygen, lipid hydroperoxides were produced in the cells. L-PhGPx was able to remove these hydroperoxides; this removal was inhibited by antisense treatment. The inhibition of L-PhGPx by the antisense oligodeoxynucleotides also resulted in increased membrane damage as measured by trypan blue dye exclusion. These data demonstrate that PhGPx expression can be manipulated by antisense techniques.     

NGF mediates the neuroprotective effect of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo: evidence from an NGF-antisense study.

Previous studies in our laboratory suggested that neuroprotective effects of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo occurred due to enhanced synthesis of nerve growth factor. The aim of the present study was to evaluate the effects of a phosphothioated NGF oligodeoxynucleotide on neuroprotection by clenbuterol in vitro and in vivo. After clenbuterol treatment (1-100 microM) an increase in nerve growth factor mRNA and protein levels (200-300% of control) was observed in primary cultures of rat cortical astrocytes. Nerve growth factor antisense oligonucleotide (0.3-1 microM for 3 days) reduced the content of nerve growth factor protein in the medium of the astrocytes concentration-dependently to 20% of control level. Nerve growth factor content in the medium of mixed hippocampal cells was reduced to 55% of sister cultures receiving the vehicle or a random control oligonucleotide. In mixed hippocampal cultures pretreated with random oligonucleotide (1 microM, 30 h), clenbuterol (10 microM) reduced the percentage of damaged neurons after glutamate exposure (0.5 mM, 1 h) to 17%. Pretreatment with nerve growth factor antisense oligonucleotide (1 microM) for 30 h before glutamate incubation blocked the protective effect of clenbuterol. In vivo, clenbuterol (0.01-0.1 mg/kg) reduced the infarct volume in a rat model of permanent focal cerebral ischemia dose-dependently. Nerve growth factor antisense oligonucleotides injected into the cortical tissue before ischemia abolished the cerebroprotective effect of clenbuterol. Our results indicate that the nerve growth factor antisense oligonucleotide presented in this study is a useful tool to investigate the effects of nerve growth factor knock down. By using the nerve growth factor antisense oligonucleotide we could demonstrate that nerve growth factor mediated the neuroprotective effects of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo.     

Peroxiredoxin 6 as an antioxidant enzyme: protection of lung alveolar epithelial type II cells from H2O2-induced oxidative stress

We evaluated the antioxidant role of peroxiredoxin 6 (Prdx6) in primary lung alveolar epithelial type II cells (AEC II) that were isolated from wild type (WT), Prdx6-/-, or Prdx6 transgenic (Tg) overexpressing mice and exposed to H(2)O(2) at 50-500 microM for 1-24 h. Expression of Prdx6 in Tg AEC II was sevenfold greater than WT. Prdx6 null AEC II exposed to H(2)O(2) showed concentration-dependent cytotoxicity indicated by decreased "live/dead" cell ratio, increased propidium iodide (PI) staining, increased annexin V binding, increased DNA fragmentation by TUNEL assay, and increased lipid peroxidation by diphenylpyrenylphosphine (DPPP) fluorescence. Compared to Prdx6 null cells, oxidant-mediated damage was significantly less in WT AEC II and was least in Prdx6 Tg cells. Thus, Prdx6 functions as an antioxidant enzyme in mouse AEC II. Prdx6 has been shown previously to reduce phospholipid hydroperoxides and we postulate that this activity is a major mechanism for the effectiveness of Prdx6 as an antioxidant enzyme.     

Phospholipid hydroperoxides are substrates for non-selenium glutathione peroxidase.

This study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity with sn-2-linolenoyl- or sn-2-arachidonoyl-phosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH. Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM(-1) min(-1)) were k(1) = 1-3 x 10(5) and k(2) = 4-11 x 10(4). In the presence of 0.36 mM GSH, apparent K(m) was 120-130 microM and apparent V(max) was 1.5-1.6 micromol/min/mg of protein. Assays with H(2)O(2) and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides. Maximal enzymatic activity was at pH 7-8. Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K(i) 4 miroM. The enzyme had no GSH S-transferase activity. Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases. The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da. These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides. Thus, this enzyme may be an important component of cellular antioxidant defense systems.     

Exposure to an Antisense Oligonucleotide Decreases Corticotropin-Releasing Factor Receptor Binding in Rat Pituitary Cultures

Abstract: Corticotropin-releasing factor (CRF) appears to integrate the endocrine, autonomic, immunologic, and behavioral responses of mammals to stress. To investigate further the role of CRF in the CNS, we have begun investigating the usefulness of "antisense knockdown" strategies directed against the CRF receptor using rat anterior pituitary gland primary cell cultures. The 15-mer antisense (5' CTG-CGG-GCG-CCG-TCC 3') and "scrambled" control (5' CGT-CCG-CGC-GCT-GCG 3') oligonucleotides were synthesized based on the rat CRF receptor sequence just downstream of the initiation codon. In each of four separate experiments, exposure to 10 µmol/L of antisense oligonucleotide for 40–67 h resulted in significant (17–36%) decreases in ¹²⁵I-ovine CRF binding to pituitary cells as compared with either control (no oligonucleotide) or 10 µmol/L of "scrambled" oligonucleotide. Moreover, compared with scrambled oligonucleotide, exposure to 10 µmol/L of antisense oligonucleotide, which produced a 22% decrease in CRF receptor binding, also resulted in a significant attenuation of the adrenocorticotrophic hormone response following a 30-min challenge with 100 pmol/L of CRF. Thus, CRF receptor antisense oligonucleotides apparently reduce functional expression of CRF receptors. This technique may be useful in studying the kinetics of CRF receptor production and the physiological functions of CRF receptors within the CNS.     

Cross-Protective Effect of Antisense Oligonucleotide Developed Against the Common 3′ NCR of Influenza A Virus Genome

The influenza A virus (IAV) has eight segmented single-stranded RNA genome containing a common and evolutionarily conserved non-coding region (NCRs) at 5′ and 3′ ends that are important for the virus replication. In this study, we designed an antisense oligonucleotide against the 3′ NCR of vital segments of the IAV genome to inhibit its replication. The results demonstrated that the co-transfection of Madine Darby Canine Kidney (MDCK) cells with the antisense oligonucleotide and the plasmids encoding the viral genes led to the down-regulation of the viral gene expression. The designed antisense molecules reduced the cytopathic effect caused by A/PR/8/34 (H1N1), A/Udorn/307/72 (H3N2), and A/New Caledonia/20/99 (H1N1) strains of IAV for almost 48 h. Furthermore, the intra-venous delivery of this oligonucleotide significantly reduced the viral titers in the lungs of infected mice and protected the mice from lethal effects of all the strains of influenza virus. The study demonstrated that the antisense oligonucleotide designed against the NCR region inhibits the expression of the viral genome. The decrease of the cytopathic effect in the MDCK cells and increase in survival of mice confirmed the reduction of virus multiplication and pathogenesis in the presence of antisense oligonucleotide. Thus, we demonstrate that a single antisense oligonucleotide is capable of providing protection against more than one strains of the IAV.     

β-Carotene as a high-potency antioxidant to prevent the formation of phospholipid hydroperoxides in red blood cells of mice

In order to investigate the antioxidant effect of β-carotene in vivo, phospholipid hydroperoxides and β-carotene isomers in red blood cells (RBC), plasma and tissue organelles were quantitatively measured after the oral administration of β-carotene (94.8% all-trans-β-carotene) to mice. Three groups of 24 mice each were fed for 1 week on a semisynthetic diet supplemented with either 0.6% or 3.0% β-carotene/diet or maintained on a control (β-carotene-unsupplemented) diet. The RBC phospholipid hydroperoxides showed a significant decrease followed by an increase of β-carotene intakes; i.e., 201, 16 and 4 pmol of phosphatidylcholine hydroperoxide/ml packed RBC, and 108, 22 and 8 pmol of phosphatidylethanolamine hydroperoxide/ml packed RBC, in the mice given the control diet, 0.6% carotene diet and 3.0% carotene diet, respectively. The RBC β-carotene increased from 14 to 43 pmol/ml packed RBC as followed by the increase of β-carotene intakes. Such a potent antioxidant effect of β-carotene as observed in RBC was not confirmed in the plasma, liver or lungs, although their β-carotene contents increased. The β-carotene ingestion increased the all-trans-β-carotene d and retinol contents in RBC, plasma, liver and lungs, but the α-tocopherol content decreased. In the β-carotene-supplemented (6 g and 30 g/kg diet) mice, cis-β-carotene content was relatively higher in the RBC (25–35% of total β-carotene) than that in plasma, liver and lungs. The present findings indicate that not only does β-carotene act as a potent antioxidant in vivo but also its antioxidant effect is very specific in the RBC phospholipid bilayers rather than in the plasma and other tissue organelles.     

Isolation and characterization of a Chinese hamster ovary cell line requiring ethanolamine or phosphatidylserine for growth and exhibiting defective phosphatidylserine synthase activity

A mutant cell line (designated M.9.1.1) requiring ethanolamine for growth was derived from Chinese hamster ovary (CHO-K1) cells using 5-bromodeoxyuridine enrichment. The ethanolamine requirement was readily replaced by 20 microM phosphatidylserine and 10 microM lysophosphatidylethanolamine. When M.9.1.1 cells were supplemented with phosphatidyl[3H]serine it was rapidly taken up, and subsequently decarboxylated to form phosphatidyl[3H]ethanolamine. The incorporation of [3H]serine into phosphatidylserine in the mutant cells was 57% of that in the parental cells. Phosphatidylethanolamine synthesis from [3H]serine in the mutant cells was 35% of that in parental cells. When M.9.1.1 cells were deprived of ethanolamine for 48 h the level of phosphatidylserine decreased 34% and the level of phosphatidylethanolamine decreased 26% compared to parental cells. At the same time the rate of turnover of phosphatidylserine was reduced to half that found in parental cells. Examination of the enzymes of phosphatidylserine metabolism indicated defective phosphatidylserine synthase activity in the mutant. When exogenous phosphatidylcholine was used as the phospholipid substrate for the reaction the apparent kinetic constants were Vmax (mutant) = 5.7 pmol/min/mg protein and Vmax (parental) = 17.5 pmol/min/mg protein. Measurement of the back reaction (ATP-independent incorporation of choline into phospholipid) gave no detectable activity in the mutant cells. The data indicate that the phosphatidylcholine-dependent synthesis of phosphatidylserine is the primary lesion in M.9.1.1.     

Lethal damage to murine L1210 cells by exogenous lipid hydroperoxides: protective role of glutathione-dependent selenoperoxidases

The effect of selenium deprivation on the viability of murine L1210 cells exposed to various exogenous lipid hydroperoxides has been investigated. Selenoperoxidase activities of cells grown for longer than 1 week in 1% serum with no added selenium [Se(-) cells] were less than 10% of the activities of selenium-satisfied controls [Se(+) cells] or selenium-repleted counterparts [Se(-/+) cells]. The enzymes measured were classical glutathione peroxidase (GPX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX). Se(-) cells exhibited a compensatory increase in catalase activity. Dye exclusion and clonal survival assays indicated that Se(-) and Se(+) cells were relatively insensitive to photochemically generated phospholipid hydroperoxides in liposomal form. However, both cell types were sensitive to liposomal cholesterol hydroperoxides, e.g., 7-hydroperoxycholesterol (7-OOH), Se(-) being much more so (LD50 approximately 10 microM) than Se(+) (LD50 approximately 75 microM). By contrast, 7-hydroxycholesterol over a comparable concentration range was minimally toxic to Se(-) and Se(+) cells. Cell killing by 7-OOH was inhibited by desferrioxamine and by butylated hydroxytoluene, suggesting that iron-mediated free radical reactions are involved. The involvement of glutathione in cytoprotection was confirmed by showing that Se(+) cells were more sensitive to 7-OOH after treating with buthionine sulfoximine, an inhibitor of GSH synthesis. Cellular detoxification of 7-OOH is provisionally attributed to PHGPX rather than GPX, since 7-OOH and other cholesterol hydroperoxides were found to be good substrates for PHGPX in a cell free system, but were unreactive with GPX.     

GRK3 mediates desensitization of CRF1 receptors: a potential mechanism regulating stress adaptation

Potential G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) mediation of homologous desensitization of corticotropin-releasing factor type 1 (CRF1) receptors was investigated in human retinoblastoma Y-79 cells. Inhibition of PKA activity by PKI(5-22) or H-89 failed to attenuate homologous desensitization of CRF1 receptors, and direct activation of PKA by forskolin or dibutyryl cAMP failed to desensitize CRF-induced cAMP accumulation. However, treatment of permeabilized Y-79 cells with heparin, a nonselective GRK inhibitor, reduced homologous desensitization of CRF1 receptors by approximately 35%. Furthermore, Y-79 cell uptake of a GRK3 antisense oligonucleotide (ODN), but not of a random or mismatched ODN, reduced GRK3 mRNA expression by approximately 50% without altering GRK2 mRNA expression and inhibited homologous desensitization of CRF1 receptors by approximately 55%. Finally, Y-79 cells transfected with a GRK3 antisense cDNA construct exhibited an approximately 50% reduction in GRK3 protein expression and an ~65% reduction in homologous desensitization of CRF1 receptors. We conclude that GRK3 contributes importantly to the homologous desensitization of CRF1 receptors in Y-79 cells, a brain-derived cell line.     

Protection of cells from oxidative stress by microsomal glutathione transferase 1

Rat liver microsomal glutathione transferase 1 (MGST1) is a membrane-bound enzyme that displays both glutathione transferase and glutathione peroxidase activities. We hypothesized that physiologically relevant levels of MGST1 is able to protect cells from oxidative damage by lowering intracellular hydroperoxide levels. Such a role of MGST1 was studied in human MCF7 cell line transfected with rat liver mgst1 (sense cell) and with antisense mgst1 (antisense cell). Cytotoxicities of two hydroperoxides (cumene hydroperoxide (CuOOH) and hydrogen peroxide) were determined in both cell types using short-term and long-term cytotoxicity assays. MGST1 significantly protected against CuOOH and against hydrogen peroxide (although less pronounced and only in short-term tests). These results demonstrate that MGST1 can protect cells from both lipophilic and hydrophilic hydroperoxides, of which only the former is a substrate. After CuOOH exposure MGST1 significantly lowered intracellular ROS as determined by FACS analysis.     

Survivin反义核酸结合位点的体外筛选及鉴定

合成 2 0mer随机寡核苷酸文库 ,与体外转录出的全长survivincRNA杂交 ,RNaseH酶切割后 ,经引物延伸、放射自显影 ,共筛选出 13个针对survivin基因的反义结合位点 (antisenseaccessiblesites ,AAS) .运用RNADraw软件分析、选定具有显著茎环结构的 4个位点 ,合成互补性反义寡核苷酸AS ODN1、AS ODN2 、AS ODN3 、AS ODN4并转染高表达survivin基因的胃癌细胞株MKN 4 5 .逆转录聚合酶链反应和Western印迹检测发现MKN 4 5细胞的survivinmRNA和蛋白水平均有显著的下降 ;MTT比色法证实 6 0 0nmol LAS ODN1～AS ODN4转染 2 4h后细胞生长受到明显抑制 ,透射电镜、annexinⅤ FITC和PI双染色流式细胞术均检测到细胞凋亡 .说明运用随机寡核苷酸文库 RNaseH酶切割与计算机分析相结合的方法 ,在体外有效筛选出survivin的反义核酸结合位点 ,其相应的反义寡核苷酸能阻断survivin基因的生物学功能 .