Induction of Superoxide Dismutases in Photobacterium Leiognathi

We investigated the induction of Cu, Zn-SOD (bacteriocuprein) and Fe-SOD in Photobacterium leiognathi DK-AI which was isolated from the light organ of the squid, Droteuthis kensaki. The induction of superoxide dismutases depended on the addition of paraquat to the medium. Induction of SOD by paraquat was attributed mostly to the bacteriocuprein by measuring of the activities of both SODs by using densitometry of isoelectrofocusing gel. When paraquat was added to the culture at various times in the early log phase of growth, the most efficient induction of the SODs. which was measured at the time of harvesting the cells (17 hours after inoculation). was observed when paroquart was added at 60 min after the inoculation. Catalase was not significantly induced by the addition of paraquat or increasing of oxygen concentration. We developed an assay of SOD by modification of a cytochrome c-xanthine oxidase method using a computer equipped absorption spectrophotometer.     

Structure-Function Relationships in Iron and Manganese Superoxide Dismutases

Using the complete sequences for MnSOD from Thermus thermophilus and for FeSOD from E. coli, structural models for both oxidized enzymes have been refined, the Mn protein to an R of 0.186 for all data between 10.0 and 1.8 Å, and the Fe protein to an R of 0.22 for data between 10.0 and 2.5 A. The results of the refinements support the presence of a solvent as a fifth ligand to Mn(III) and Fe(III) and a coordination geometry that is close to trigonal bipyramidal. The putative substrate-entry channel is comprised of residues from both subunits of the dimer; several basic residues that are conserved may facilitate approach of O^?₂, while other conserved residues maintain interchain packing interactions. Analysis of the azide complex of Fe(III) dismutase suggests that during turnover O^?₂ binds to the metal at a sixth coordination site without displacing the solvent ligand. Because crystals reduced with dithionitc show no evidence for displacement of the protein ligands, the redox-linked proton acceptor (C. Bull and J.A. Fee (1985), Journol of the American Chemistry Society 107, 3295–3304) is unlikely to be one of the histidines which bind the metal ion. Structural, kinetic, titration, and spectroscopic data can be accommodated in a mechanistic scheme which accounts for the differential titration behaviour of the Fe(II1) and Fc(II) enzymes at neutral and high pH.     

Superoxide Dismutases of Escherichia coli: Intracellular Localization and Functions

Escherichia coli B contains two superoxide dismutases which differ with respect to their localization within the cell, the nature of their prosthetic metals, their responses to changes in (p)O(2), and their functions. One of these enzymes, which was liberated from the cells by osmotic shock and which was therefore presumed to be localized in the periplasmic space, is an iron-containing superoxide dismutase. The amount of this iron enzyme did not vary in response to changes in (p)O(2) during growth. In contrast, the other superoxide dismutase was not solubilized by osmotic shock, was a mangano-protein, and was found in greater amounts in cells which had been grown at high (p)O(2). E. coli, which had low levels of the iron-enzyme and high levels of the mangano-enzyme, as a consequence of growth in iron-deficient aerated medium, was killed by exposure to an exogenous flux of O(2) (-) which was generated either photochemically or enzymatically. The addition of bovine superoxide dismutase to the suspending medium protected these cells against this stress. On the other hand, E. coli, which had high levels of the iron-enzyme and low levels of the mangano-enzyme, as a consequence of growth in iron-rich anaerobic medium, was resistant to exogeneous O(2) (-). On the basis of these and of previously reported results (4a, Yost, F. J. and I. Fridovich, J. Biol. Chem., 1973, in press), it appears that the iron superoxide dismutase, of the periplasmic space, serves as a defense against exogenous O(2) (-), whereas the mangano-superoxide dismutase, in the matrix of these cells, serves to counter the toxicity of endogenous O(2) (-).     

Superoxide Dismutases: II. Purification and Quantitative Relationship with Water-soluble Protein in Seedlings

Superoxide dismutase was purified from pea (Pisum sativum L., cv. Wando) seeds and corn (Zea mays L., cv. Michigan 500) seedlings. The purified pea enzyme eluting as a single peak from gel exclusion chromatography columns contained the three electrophoretically distinct bands of superoxide dismutase characterizing the crude extract. The purified corn enzyme eluted as the same peak as the pea enzyme, and contained five of the seven active bands found in the crude extract. The similar molecular weights and the cyanide sensitivities of these bands indicated that they are probably isozymes of a cupro-zinc superoxide dismutase. One of the remaining corn bands was shown to be a peroxidase.     

Biosynthesis and Cellular Distribution of the Two Superoxide Dismutases of Dactylium dendroides

The synthesis and subcellular localization of the two superoxide dismutases of Dactylium dendroides were studied in relation to changes in copper and manganese availability. Cultures grew normally at all medium copper concentrations used (10 nM to 1 mM). In the presence of high (10 μM) copper, manganese was poorly absorbed in comparison to the other metals in the medium. However, cells grown at 10 nM copper exhibited a 3.5-fold increase in manganese content, while the concentration of the other metals remained constant. Cultures grown at 10 nM copper or more had 80% Cu/Zn enzyme and 20% mangani enzyme; the former was entirely in the cytosol, and the latter was mitochondrial. Removal of copper from the medium resulted in decreased Cu/Zn superoxide dismutase synthesis with a concomitant increase in the mangani enzyme such that total cellular superoxide dismutase activity remained constant. The mangani enzyme in excess of the 20% was present in the non-mitochondrial fraction. The mitochondria, therefore, show no variability with respect to superoxide dismutase content, whereas the soluble fraction varies from 100 to 13% Cu/Zn superoxide dismutase. Copper-starved cells that were synthesizing predominantly mangani superoxide dismutase could be switched over to mostly Cu/Zn superoxide dismutase synthesis by supplementing the medium with copper during growth. Immunoprecipitation experiments suggest that the decrease in Cu/Zn activity at low copper concentration is a result of decreased synthesis of that protein rather than the production of an inactive apoprotein.     

Comparative Anti-Inflammatory Activity of Different Superoxide Dismutases and Liposomal Sod in Ischemia

Comparison of superoxide dismutases from different sources with respect to biological activity in the rat tourniquet poditis model shows that anti-ischemic activity is very variable although all the enzymes have the same specific enzymic activity. Both bovine Cu-SOD and E. coli Mn-SOD have excellent properties whereas yeast Cu-SOD and the homologous rat Cu-SOD show zero activity. The results confirm earlier demonstrations that (1) “All superoxide dismutases are equal but some are more equal than others”, (2) at the dose levels used (compatible with possible clinical use) homologous enzyme is inefficient and hence human Cu-SOD may not be effective in humans, (3) liposomal encapsulation of bovine Cu-SOD greatly enhances biological efficacity, provides a slow release mechanism of the enzyme and provides a powerful drug for the treatment of ischemic injury.     

Anaerobic Inductions of Active Forms of Superoxide Dismutases in Escherzchia Coli

Escherichio coli growing anaerobically respond to NO^?₃ with a ～ 3-fold induction of active FeSOD and a ～ 5.5-fold induction of an inactive, but activatable form of MnSOD (pro-MnSOD). Paraquat, which mediates anaerobic electron flow to NO₃-, increased the induction of pro-MnSOD to ~ 25-fold. Strains with defects in the SOD genes or which lacked nitrate reductase activity failed to accumulate active or pro-forms of SODS in response to NO₃-± PQ^{+ +}. Diamide caused anaerobic induction of active MnSOD and this effect was also observed in a glutathione-negative strain. These inductions required de novo synthesis of protein, even when cell content of pro-MnSOD had been elevated by exposure to NO₃-+ PQ⁺⁺ prior to addition of diamide.

These results indicate that oxidation of a cell component increases biosynthesis of the SOD gene product and this postulated oxidation can be caused by terminal electron acceptors, such as dioxygen or NO₃-. In addition, it appears that insertion of the correct metal can be rate-limiting, leading to competition by other metals and to the accumulation of inactive, incorrectly substituted pro-forms. Metal insertion may be dependent upon the valence of the metal, which may be influenced, in turn, by the redox status of the cells. Diamide and redox active agents such as ferricyanide may thus allow anaerobic production of active MnSOD by favoring the production of a complexed form of Mn(III) which can compete favorably with other metal cations for the active site of nascent MnSOD.     

鸡冠花幼苗热胁迫耐性与其SOD之间的关联

为探讨鸡冠花热胁迫耐性与其SOD之间的关联,选用耐热品种Variety-Centrury Green 10叶期幼苗为试材,用二乙基二硫代氨基甲酸钠(DDTC)进行48 h预处理,之后在45℃人工培养箱中进行热胁迫处理,观察其外观形态.结果表明,20 mmol/L DDTC预处理显著抑制叶片SOD活性,明显减弱鸡冠花的热胁迫耐性,表现为幼苗热胁迫耐受时间显著缩短,弯颈、死亡率明显提高.在自然状态下,叶片中有1种MnSOD、1种Cu/ZnSOD和3种FeSOD;迁移率大小依次为Cu/ZnSODFeSODMnSOD;谱带强弱依次为FesOD>Cu/znSOD>MnSOD.经热胁迫处理后,各种SOD同工酶条带亮度均呈现不同程度的增强—减弱的变化趋势,并诱导产生了1条新的Cu/Zn-SOD条带,与此同时MnSOD条带最先消失.由此推测,鸡冠花品种间耐热性差异与其SOD活性相关,与胁迫强度相对应,同时也与Cu/ZnSOD2的诱导产生相关联. 表现为幼苗热胁迫耐受时间显著缩短,弯颈、死亡率明显提高.在自然状态下,叶片中有1种MnSOD、1种Cu/ZnSOD和3种FeSOD;迁移率大小依次为Cu/ZnSODFeSODMnSO ;谱带强弱依次为FesOD>Cu/znSOD>MnSOD.经热胁迫处理后,各种SOD同工酶条带亮度均呈现不同程度的增强一减弱的变化趋势,并诱导产生了1条新的Cu/Zn-SOD条带,与此同时MnSOD条带最先消失.由此推测,鸡冠花品种间耐热性差异与其SOD活性相关,与胁迫强度相对应,同时也与Cu/ZnSOD2的诱导产生     

The response of Paracoccidioides spp. to nitrosative stress

Paracoccidioidomycosis (PCM) is an endemic disease in Latin America caused by species belonging to the genus Paracoccidioides. During infection, immune cells present a variety of defense mechanisms against pathogens. One of these defensive strategies is the production and release of nitric oxide (NO) and S-nitroso thiols (e.g., S-nitrosoglutathione, GSNO), which produce reactive nitrogen species (RNS). This results in damage to DNA and membranes, inhibition of respiration and inactivation of cellular enzymes. In response to nitrosative stress, human pathogenic fungi possess defense mechanisms to prevent the adverse effects of NO, which helps them survive during initial contact with the host immune system. To understand how Paracoccidioides spp. respond to nitrosative stress, we conducted this study to identify genes and proteins that might contribute to this response. The results of proteomic analysis demonstrated that nitrosative stress induced a reduction in the expression of proteins related to the mitochondrial electron transport chain. This hypothesis was supported by the reduced mitochondrial activity observed in the presence of GSNO. Additionally, lipids and branched chain amino acid metabolism enzymes were altered. The role played by enzymes acting in oxidative stress in the RNS response was remarkable. This interface among enzymes acting in both stress responses was confirmed by using a RNA approach to silence the ccp gene in Paracoccidioides. It was observed that mutants with low expression of the ccp gene were more sensitive to nitrosative stress.     

Micronutrient Deficiency Influences Plant Growth and Activities of Superoxide Dismutases in Narrow-leafed Lupins

The effect of copper (Cu), zinc (Zn) or manganese (Mn) deficiencyon the growth and activity of superoxide dismutase (SOD) formswas investigated in seedlings of narrow-leafed lupins (LupinusangustifoliusL.). Plants grown without Zn developed Zn deficiencysymptoms 24 d after sowing (DAS), and those grown without Mnshowed Mn deficiency symptoms 31 DAS. However, plants grownwithout Cu did not show visible leaf symptoms. Shoot dry weightwas decreased by Zn and Mn deficiency 24 DAS, and by Cu deficiency31 DAS. Soluble protein concentration was reduced considerablyby Zn deficiency 24 DAS, but was not affected by Cu deficiencyuntil 31 DAS. In contrast, soluble protein concentration inMn-deficient plants was higher than in control plants 31 DAS.Shoot concentration of micronutrients which were not suppliedto plants decreased significantly, with a simultaneous increasein concentration of one or more of the other nutrients analysed.The activities of total SOD, MnSOD and Cu/ZnSOD on a fresh weightbasis declined drastically in -Cu and -Zn plants 24 DAS. Onthe contrary, the activities of total SOD and Cu/ZnSOD on eithera fresh weight or soluble protein basis increased markedly in-Mn plants 24 DAS, and MnSOD activity increased significantlyin these plants 31 DAS. It was concluded that micronutrientdeficiency (Cu, Zn or Mn) altered the activities of SOD formsdepending on the kind and severity of the deficiency stress.Manipulation of the capacity of plants to tolerate oxidativestress may influence their capacity to tolerate micronutrientdeficiency.Copyright 1999 Annals of Botany Company. Copper,Lupinus angustifolius, manganese, deficiency, superoxide dismutase, zinc.     

Anti-Inflammatory Activity of Superoxide Dismutases: Studies on Adjuvant Induced Polyarthritis in Rats

The anti-arthritic activities of various superoxide dismutases and of liposomal bovine Cu-SOD have been compared in the adjuvant induced Lewis Inbred Rat model. Various approaches, including plethysmometric measurements, red cell sedimentation rates, while cell counts, levels of IgA and IgG immunoglobulins and scoring by visual, radiographic and scintigraphic techniques all concord in a demonstration of different activities for different SODs. The most efficient are liposomal bovine Cu-SOD and E. coli Mn-SOD, a moderate activity being shown by free bovine Cu-SOD. Poor or zero results are obtained with human Mn-SOD, human Cu-SOD or the homologous rat Cu-SOD.     

Anti-Inflammatory Activity of Superoxide Dismutases: Inhibition of Carrageenan Induced Edema in Rats

Eighteen different superoxide dismutases from procaryote, plant, fish, bird and mammalian species have been tested for anti-inflammatory activity in the rat paw pad carrageenan-induced inflammation model Very large differences in activity are observed. Homologous rat Cu-SOD is not active and indeed shows slight pro-inflammatory activity. The different SODs have different iso-electric values, different metal; (Cu, Mn or Fe) at the active centre, different molecular weights and different circulation lifetimes Biological activity is a function of amino acid sequence rather than of such secondary parameters.     

Anti-Inflammatory Activity of Superoxide Dismutases: Inhibition of Carrageenan Induced Edema in Rats

Eighteen different superoxide dismutases from procaryote, plant, fish, bird and mammalian species have been tested for anti-inflammatory activity in the rat paw pad carrageenan-induced inflammation model Very large differences in activity are observed. Homologous rat Cu-SOD is not active and indeed shows slight pro-inflammatory activity. The different SODs have different iso-electric values, different metal; (Cu, Mn or Fe) at the active centre, different molecular weights and different circulation lifetimes Biological activity is a function of amino acid sequence rather than of such secondary parameters.     

Anti-Inflammatory Activity of Superoxide Dismutases: Inhibition of Adriamycin Induced Edema in Rats

Various superoxide dismutases from different sources, containing Cu, Mn or Fe at the active centre, have been examined with respect to anti-inflammatory activity in a model using adriamycin-induced edema in rats. Very large differences in efficiency are observed, the most active being E. coli Mn-SOD and bovine Cu-SOD. TheFe-SOD from E coli is active whereas P. leiognathiFe-SODisnot. Human Mn-SOD shows no significant activity and homologous rat Cu-SOD is totally inactive. Yeast Cu-SOD shows proinflammatory properties. Anti-inflammatory activity is not a function of molecular weight or circulation life-time.     

Oversynthesis of Riboflavin in the Yeast Pichia guilliermondii is Accompanied by Reduced Catalase and Superoxide Dismutases Activities

Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. Under iron deprivation conditions, Pichia guilliermondii cells increase production of riboflavin and malondialdehyde and the formation of protein carbonyl groups, which reflect increased intracellular content of reactive oxygen species. In this study, we found that P. guilliermondii iron deprived cells showed dramatically decreased catalase and superoxide dismutase activities. Previously reported mutations rib80, rib81, and hit1, which affect repression of riboflavin synthesis and iron accumulation by iron ions, caused similar drops in activities of the mentioned enzymes. These findings could explain the previously described development of oxidative stress in iron deprived or mutated P. guilliermondii cells that overproduce riboflavin. Similar decrease in superoxide dismutase activities was observed in iron deprived cells in the non-flavinogenic yeast Saccharomyces cerevisiae.     

Characteristic Isozyme Spectra of Anionic Peroxidases and Superoxide Dismutases in Callus Cultures of Larix sibirica Ledeb. and Larix gmelinii Rupr. Rupr.

Multiple molecular forms of anionic peroxidase (AP) and superoxide dismutase (SOD) were investigated in the callus lines of Larix sibirica and L. gmelinii. Eight distinct patterns of the AP spectra were discerned among 13 investigated lines. The spectra of SOD molecular forms were similar in all lines under investigation, although the lines were obtained from two Larix species and the calli were of different origin. Fe-containing SOD was for the first time described in the Larix isoenzyme spectrum. The authors conclude that the SOD isozyme spectra in dedifferentiated cells of L. sibirica and L. gmelinii are more stable than the isoperoxidase spectra.     

Identification and Functional Analysis of Three Isoforms of Bovine BST-2

Human BST-2 (hBST-2) has been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. Orthologues of BST-2 have been identified in several species, including human, monkeys, pig, mouse, cat and sheep. All have been reported to possess antiviral activity. Duplication of the BST-2 gene has been observed in sheep and the paralogues are referred to as ovine BST-2A and BST2-B, although only a single gene corresponding to BST-2 has been identified in most species. In this study, we identified three isoforms of bovine BST-2, named bBST-2A1, bBST-2A2 and bBST-2B, in bovine cells treated with type I interferon, but not in untreated cells. Both bBST-2A1 and bBST-2A2 are posttranslationally modified by N-linked glycosylation and a GPI-anchor as well as hBST-2, while bBST-2B has neither of these modifications. Exogenous expression of bBST-2A1 or bBST-2A2 markedly reduced the production of bovine leukemia virus and vesicular stomatitis virus from cells, while the antiviral activity of bBST-2B was much weaker than those of bBST-2A1 and bBST-2A2. Our data suggest that bBST-2A1 and bBST-2A2 function as part of IFN-induced innate immunity against virus infection. On the other hand, bBST-2B may have a different physiological function from bBST-2A1 and bBST-2A2.     

脂肪酸组分分析在不动杆菌鉴定中的应用

以不动杆菌属(Acinetobacter)中的A. baumannii、A. haemolyticus、A. johnsonii、A. junii、A. lwoffii模式菌株为参比菌株,对中国农业微生物菌种保藏管理中心(ACCC)所保藏的6株不动杆菌进行脂肪酸组分分析及16S rRNA基因系统进化分析.结果表明,利用脂肪酸分析可将6株菌完全准确地鉴定到属的水平上,这一点与16S rRNA基因分析结果一致;在种的水平上,16S rRNA基因进化分析与脂肪酸组分分析的结果可互为补充,相互印证.同时,通过对不动杆菌部分模式菌株及供试菌株的脂肪酸组分分析,报道了不动杆菌的特征性脂肪酸为C18:1 w9c、C16:00和C16:1w7c/C16:1w6c.