Peroxynitrite reductase activity of bacterial peroxiredoxins

Nitric oxide (NO) is present in soil and air, and is produced by bacteria, animals and plants. Superoxide (O2-) arises in all organisms inhabiting aerobic environments. Thus, many organisms are likely to encounter peroxynitrite (OONO-), a product of NO and O2- that forms at near diffusion-limited rates, and rapidly decomposes upon protonation through isomerization to nitrate (NO3-; ref. 1) while generating hydroxyl radical (*OH) and nitrogen dioxide radical (*NO2) (refs 2, 3), both more reactive than peroxynitrite's precursors. The oxidative, inflammatory, mutagenic and cytotoxic potential (ref. 4) of peroxynitrite contrasts with the antioxidant, anti-inflammatory and tissue-protective properties ascribed to NO itself. Thus, the ability of cells to cope with peroxynitrite is central in determining the biological consequences of NO production. We considered whether cells might be equipped with enzymes to detoxify peroxynitrite. Peroxiredoxins have been identified in most genomes sequenced, but their functions are only partly understood. Here we show that the peroxiredoxin alkylhydroperoxide reductase subunit C (AhpC) from Salmonella typhimurium catalytically detoxifies peroxynitrite to nitrite fast enough to forestall the oxidation of bystander molecules such as DNA. Results are similar with peroxiredoxins from Mycobacterium tuberculosis and Helicobacter pylori. Thus, peroxynitrite reductase activity may be widespread among bacterial genera.     

几种脊椎动物红细胞血型的比较研究

采用两种方法对脊椎动物各纲的代表动物小白鼠、家兔、家鸽、蟾蜍、鲫鱼的红细胞血型进行了研究 .方法一 :采用人类抗A、抗B标准血清与上述动物的红细胞进行反应 ,结果均未出现凝集现象 ,表明 :这几种动物的红细胞膜上 ,没有与人类抗A、抗B标准血清起反应的A凝集原和B凝集原 .方法二 :采用同种动物的血清与同种动物不同个体的红细胞进行反应 ,结果 :上述几种动物的红细胞与同种内不同个体的血清均不发生凝集反应 .     

NO在负载型金属氧化物催化剂上的氧化反应机理

研究了γ- Al2 O3负载的金属氧化物催化剂对 NO催化氧化性能。实验发现 ,30 0°C下催化剂活性顺序为 Mn>Cr>Co>Cu>Fe>Ni>Zn,P-型金属氧化物对 NO的氧化活性较强。对 Mn O2这种具有代表性的 P-型金属氧化物催化剂 ,提出了其 NO催化氧化的反应机理和动力学模型。     

B→O blood conversion

To maintain a constant supply of “universal blood type”,or group O red blood cells,has benefit in specialized transfusion condition.In this study, α-galactosidase cDNA was cloned from Catimor coffee bean grown in Hainan island,China,by the RT-PCR method.We have constructed a vector for α-galactosidase cDNA expression and transferred α-galactosidase cDNA into Pichia pastoris GS115 cells by electroporation.The recombinant α-galactosidase(r-α-GalE)was purified by cation ion exchange chromatography.After studying the biochemical characters of r-α-GalE we have succeeded in converting human erythrocytes from group B to group O.The animal experiment showed that transfusion of enzymetically converted group O red blood cells(ECORBC)was perfectly safe.     

S-glutathionylation uncouples eNOS and regulates its cellular and vascular function

Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular function, and can generate both nitric oxide (NO) and superoxide (O(2)(?-)), which are key mediators of cellular signalling. In the presence of Ca(2+)/calmodulin, eNOS produces NO, endothelial-derived relaxing factor, from l-arginine (l-Arg) by means of electron transfer from NADPH through a flavin containing reductase domain to oxygen bound at the haem of an oxygenase domain, which also contains binding sites for tetrahydrobiopterin (BH(4)) and l-Arg. In the absence of BH(4), NO synthesis is abrogated and instead O(2)(?-) is generated. While NOS dysfunction occurs in diseases with redox stress, BH(4) repletion only partly restores NOS activity and NOS-dependent vasodilation. This suggests that there is an as yet unidentified redox-regulated mechanism controlling NOS function. Protein thiols can undergo S-glutathionylation, a reversible protein modification involved in cellular signalling and adaptation. Under oxidative stress, S-glutathionylation occurs through thiol-disulphide exchange with oxidized glutathione or reaction of oxidant-induced protein thiyl radicals with reduced glutathione. Cysteine residues are critical for the maintenance of eNOS function; we therefore speculated that oxidative stress could alter eNOS activity through S-glutathionylation. Here we show that S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in O(2)(?-) generation primarily from the reductase, in which two highly conserved cysteine residues are identified as sites of S-glutathionylation and found to be critical for redox-regulation of eNOS function. We show that eNOS S-glutathionylation in endothelial cells, with loss of NO and gain of O(2)(?-) generation, is associated with impaired endothelium-dependent vasodilation. In hypertensive vessels, eNOS S-glutathionylation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents, which reverse this S-glutathionylation. Thus, S-glutathionylation of eNOS is a pivotal switch providing redox regulation of cellular signalling, endothelial function and vascular tone.     

有氧条件下Au/CeO2/Al2O3催化还原NO的研究

NO是形成光化学烟雾、生成酸雨和破坏大气臭氧层的前驱气体之一，消除NO巳成为近几年国内外催化界的研究热点，Cu0-ZSM-5是国内外研究最成熟的催化剂，但实用性差，本研究以HAuCl4为前驱体，沉积-沉淀法制备Au/CeO2/Al2O3催化剂，用X-射线衍射（XRD）进行表征，考察了加入CeO2对Au/CeO2/Al2O3催化活性的影响。结果表明：Al2O3、CeO2/Al2O3表面的Au晶粒小，分散度高，Au/Al2O3的deNOx活性较低，而含金复合催化剂Au/CeO2/Al2O3的活性高于Au/Al2O3及Cu-ZSM-5。     

GILT is a critical host factor for Listeria monocytogenes infection

Listeria monocytogenes is a gram-positive, intracellular, food-borne pathogen that can cause severe illness in humans and animals. On infection, it is actively phagocytosed by macrophages; it then escapes from the phagosome, replicates in the cytosol, and subsequently spreads from cell to cell by a non-lytic mechanism driven by actin polymerization. Penetration of the phagosomal membrane is initiated by the secreted haemolysin listeriolysin O (LLO), which is essential for vacuolar escape in vitro and for virulence in animal models of infection. Reduction is required to activate the lytic activity of LLO in vitro, and we show here that reduction by the enzyme gamma-interferon-inducible lysosomal thiol reductase (GILT, also called Ifi30) is responsible for the activation of LLO in vivo. GILT is a soluble thiol reductase expressed constitutively within the lysosomes of antigen-presenting cells, and it accumulates in macrophage phagosomes as they mature into phagolysosomes. The enzyme is delivered by a mannose-6-phosphate receptor-dependent mechanism to the endocytic pathway, where amino- and carboxy-terminal pro-peptides are cleaved to generate a 30-kDa mature enzyme. The active site of GILT contains two cysteine residues in a CXXC motif that catalyses the reduction of disulphide bonds. Mice lacking GILT are deficient in generating major histocompatibility complex class-II-restricted CD4(+) T-cell responses to protein antigens that contain disulphide bonds. Here we show that these mice are resistant to L. monocytogenes infection. Replication of the organism in GILT-negative macrophages, or macrophages expressing an enzymatically inactive GILT mutant, is impaired because of delayed escape from the phagosome. GILT activates LLO within the phagosome by the thiol reductase mechanism shared by members of the thioredoxin family. In addition, purified GILT activates recombinant LLO, facilitating membrane permeabilization and red blood cell lysis. The data show that GILT is a critical host factor that facilitates L. monocytogenes infection.     

在石墨炉内石墨探针表面上的原子化机理研究XI.硝酸铋的原子化机理

研究了硝酸铋在石墨炉内石墨探针表面上的原子化机理，结果表明．硝酸铋首先分解为氧化铋，后者被碳还原生成铋，铋再蒸发进入气相，经历二聚体，尔后分解为气态铋原子。     

Pt@g-C_3N_4/CeO_2 photocatalyst for the remediation of low concentration NO_x at room temperature

Pt@g-C_3N_4/CeO_2 photocatalyst was prepared by calcination of g-C_3N_4/CeO_2 mixture followed by impregnating Pt. Under the irradiation of visible light with the presence of water vapor at room temperature, Pt@g-C_3N_4/CeO_2 showed significant performance for remediation of NO_x. The deNO_x mechanism was studied by modified in-situ FTIR. The results proved that NO was prone to adsorb on the surface of Pt@CeO_2 and oxidized to nitrite for storage. Then, the photoinduced hydrogen on Pt@g-C_3N_4 component could diffuse into the vicinity of nitrite through the spillover effect of Pt and reacted with nitride to produce N_2 and H_2O.     

Hereditary spherocytosis associated with deletion of human erythrocyte ankyrin gene on chromosome 8

Hereditary spherocytosis (HS) is one of the most common hereditary haemolytic anaemias. HS red cells from both autosound dominant and recessive variants are spectrin-deficient, which correlates with the severity of the disease. Some patients with recessive HS have a mutation in the spectrin alpha-2 domain (S.L.M. et al., unpublished observations), and a few dominant HS patients have an unstable beta-spectrin that is easily oxidized, which damages the protein 4.1 binding site and weakens spectrin-actin interactions. In most patients, however, the cause of spectrin deficiency is unknown. The alpha- and beta-spectrin loci are on chromosomes 1 and 14 respectively. The only other genetic locus for HS is SPH2, on the short arm of chromosome 8 (8p11). This does not correspond to any of the known loci of genes for red cell membrane proteins including protein 4.1 (1p36.2-p34), the anion exchange protein (AE1, band 3; 17q21-qter), glycophorin C (2q14-q21), and beta-actin (7pter-q22). Human erythrocyte ankyrin, which links beta-spectrin to the anion exchange protein, has recently been cloned. We now show that the ankyrin gene maps to chromosome 8p11.2, and that one copy is missing from DNA of two unrelated children with severe HS and heterozygous deletions of chromosome 8 (del(8)(p11-p21.1)). Affected red cells are also ankyrin-deficient. The data suggest that defects or deficiency or ankyrin are responsible for HS at the SPH2 locus.     

Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide

The molecular machinery responsible for the generation of transport carriers moving from the Golgi complex to the plasma membrane relies on a tight interplay between proteins and lipids. Among the lipid-binding proteins of this machinery, we previously identified the four-phosphate adaptor protein FAPP2, the pleckstrin homology domain of which binds phosphatidylinositol 4-phosphate and the small GTPase ARF1. FAPP2 also possesses a glycolipid-transfer-protein homology domain. Here we show that human FAPP2 is a glucosylceramide-transfer protein that has a pivotal role in the synthesis of complex glycosphingolipids, key structural and signalling components of the plasma membrane. The requirement for FAPP2 makes the whole glycosphingolipid synthetic pathway sensitive to regulation by phosphatidylinositol 4-phosphate and ARF1. Thus, by coupling the synthesis of glycosphingolipids with their export to the cell surface, FAPP2 emerges as crucial in determining the lipid identity and composition of the plasma membrane.     

Type VI secretion delivers bacteriolytic effectors to target cells

Peptidoglycan is the major structural constituent of the bacterial cell wall, forming a meshwork outside the cytoplasmic membrane that maintains cell shape and prevents lysis. In Gram-negative bacteria, peptidoglycan is located in the periplasm, where it is protected from exogenous lytic enzymes by the outer membrane. Here we show that the type VI secretion system of Pseudomonas aeruginosa breaches this barrier to deliver two effector proteins, Tse1 and Tse3, to the periplasm of recipient cells. In this compartment, the effectors hydrolyse peptidoglycan, thereby providing a fitness advantage for P. aeruginosa cells in competition with other bacteria. To protect itself from lysis by Tse1 and Tse3, P. aeruginosa uses specific periplasmically localized immunity proteins. The requirement for these immunity proteins depends on intercellular self-intoxication through an active type VI secretion system, indicating a mechanism for export whereby effectors do not access donor cell periplasm in transit.     

Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor

Endothelium-derived relaxing factor (EDRF) is a labile humoral agent which mediates the action of some vasodilators. Nitrovasodilators, which may act by releasing nitric oxide (NO), mimic the effect of EDRF and it has recently been suggested by Furchgott that EDRF may be NO. We have examined this suggestion by studying the release of EDRF and NO from endothelial cells in culture. No was determined as the chemiluminescent product of its reaction with ozone. The biological activity of EDRF and of NO was measured by bioassay. The relaxation of the bioassay tissues induced by EDRF was indistinguishable from that induced by NO. Both substances were equally unstable. Bradykinin caused concentration-dependent release of NO from the cells in amounts sufficient to account for the biological activity of EDRF. The relaxations induced by EDRF and NO were inhibited by haemoglobin and enhanced by superoxide dismutase to a similar degree. Thus NO released from endothelial cells is indistinguishable from EDRF in terms of biological activity, stability, and susceptibility to an inhibitor and to a potentiator. We suggest that EDRF and NO are identical.     

NO and H2O2 induced by Verticillium dahliae toxins and its influence on the expression of GST gene in cotton suspension cells

Nitric oxide （NO） and hydrogen peroxide （H2O2） have been shown to be important signaling molecules that participate in the regulation of several physiological processes. In particular, they have significant role in plant resistance to pathogens by contributing to induction defense genes. Here, whether NO and H2O2 participate in the resistance responses against Verticillium dahliae toxins （VD-toxins） and their effects on the expression of GSTgene are studied. The results reveal that NO and H2O2 are produced as part of a complex network of signals that respond to VD-toxins and may converge to function both synergistically and independently by inducing resistant responses. GSTgene is potentially involved in the resistance mechanism in the cotton suspension cells. NO induces the expression of GSTgene independently of H2O2. H2O2 may be a more potent signal in the resistance responses against VD-toxins.     

Energy source of flagellar type III secretion

Bacterial flagella contain a specialized secretion apparatus that functions to deliver the protein subunits that form the filament and other structures to outside the membrane. This apparatus is related to the injectisome used by many gram-negative pathogens and symbionts to transfer effector proteins into host cells; in both systems this export mechanism is termed 'type III' secretion. The flagellar secretion apparatus comprises a membrane-embedded complex of about five proteins, and soluble factors, which include export-dedicated chaperones and an ATPase, FliI, that was thought to provide the energy for export. Here we show that flagellar secretion in Salmonella enterica requires the proton motive force (PMF) and does not require ATP hydrolysis by FliI. The export of several flagellar export substrates was prevented by treatment with the protonophore CCCP, with no accompanying decrease in cellular ATP levels. Weak swarming motility and rare flagella were observed in a mutant deleted for FliI and for the non-flagellar type-III secretion ATPases InvJ and SsaN. These findings show that the flagellar secretion apparatus functions as a proton-driven protein exporter and that ATP hydrolysis is not essential for type III secretion.     

还生口服液抗癌作用机制的研究

报道了还生口服液对荷瘤小鼠（Ｓ１８０、ＥＡＣ、Ｌ６１５）癌细胞膜、红细胞膜Ｎａ＋，Ｋ＋－ＡＴＰａｓｅ活性的影响和对荷瘤小鼠红细胞膜免疫功能的影响。结果表明，还生口服液可显著抑制癌细胞膜、荷瘤小鼠红细胞膜Ｎａ＋，Ｋ＋－ＡＴＰａｓｅ的活性，同时可显著提高荷瘤小鼠红细胞膜的免疫功能，可使红细胞免疫复合物花环率（ＲＦＩＲ）降低，而使红细胞Ｃ３ｂ受体花环率（ＲＢＣ—Ｃ３ｂＲＲ）、红细胞Ｃ３ｂ受体花环促进率（ＲＦＥＲ）显著提高。本文结果揭示，还生口服液抗癌作用机制有部分是通过抑制癌细胞膜，荷瘤机体红细胞膜Ｎａ＋，Ｋ＋－ＡＴＰａｓｅ的活性，提高荷瘤机体红细胞免疫功能完成的。     

盐酸埃克替尼通过激活蛋白激酶C诱导Tca8113细胞表达iNOS和凋亡

体外培养Tca8113细胞,随机分为4组,(1)对照组;(2)埃克替尼处理组(20μmo1/L);(3)PKC抑制剂Ro31-8220(3μmol/L)处理组;(4)埃克替尼+Ro31-8220组(20μmo1/L Icotinib,3μmol/L Ro31-8220),不同干预因素处理细胞4h.Western blot检测不同处理组的胞膜内的PKC-α的表达水平,iNOS ELISA检测试剂盒测定细胞的iNOS含量,Griess方法测定Tca8113细胞产生的NO水平,用DNA fragmentation检测Tca8113细胞的凋亡情况.结果表明埃克替尼组DNA fragmentation细胞凋亡及上清液的iNOS,NO较对照组均明显升高;埃克替尼组细胞膜的PKC-α蛋白表达量明显增加.用埃克替尼与PKC的抑制剂Ro31-8220共同处理Tca8113细胞后,胞膜的PKC-α,iNOS的蛋白表达水平及产生的NO能被Ro31-8220显著抑制,且PKC抑制剂Ro31-8220能逆转埃克替尼引起的Tca8113细胞凋亡.埃克替尼能诱导Tca8113细胞iNOS表达和NO生成增加并能诱导Tca8113细胞凋亡;埃克替尼能促进细胞膜内的PKC-α表达增加;埃克替尼诱导的Tca8113细胞iNOS表达和凋亡与PKC有关.     

二甲基醚发动机HCCI燃烧与排放的计算

构建了可应用于均质充量压燃（HCCI）发动机的二甲基醚（DME）详细化学反应动力学M燃烧模型,该模型包含97种物种和457个基元反应．拓展M模型的应用范围,分析了DME发动机HCCI条件下关键基元反应和重要物种随曲轴转角的变化关系,获得了DME氧化反应的主要历程．研究了NOx的生成机理,结果表明NOx排放中NO生成量达到最大值后出现“冻结”现象,NO2与N2O最终排放浓度极少且受缸内温度影响不大．随着缸内温度的增加,NOx排放中NO所占比例逐渐增加．基于化学反应速率及敏感度分析,得到了DME发动机HCCI燃烧的NO排放主要受扩大Zeldovich机理和N2O途径控制．     

硒对NO诱导的内皮细胞损伤的抑制机制研究

用外源性NO供体S－亚硝基谷胱甘肽（GSNO）处理人脐静脉内皮细胞系ECV－304细胞，研究其对细胞的损伤机制，并探讨硒在这一过程中的保护作用，通过MTT法测定细胞存活率，分光光度法[测定细胞LDH漏出率及细胞脂质过氧化水平，采用荧光标记技术研究细胞膜流动性变化，结果表明，NO可引起细胞脂质过氧化水平升高，细胞膜流动性下降，导致ECV－304细胞损伤，其作用具有浓度效应，细胞内硒可通过抑制细胞脂质过氧化水平及细胞膜流动性变化从而抑制NO诱导的细胞损伤。