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1.
The cooCTJ gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni(2+) transport assay was used to monitor uptake and accumulation of (63)Ni(2+) into R. rubrum and to observe the effect of mutations in the cooC, cooT, and cooJ genes on (63)Ni(2+) transport and accumulation. Cells grown either in the presence or absence of CO transported Ni(2+) with a K(m) of 19 +/- 4 microM and a V(max) of 310 +/- 22 pmol of Ni/min/mg of total protein. Insertional mutations disrupting the reading frame of the cooCTJ genes, either individually or all three genes simultaneously, transported Ni(2+) the same as wild-type cells. The nickel specificity for transport was tested by conducting the transport assay in the presence of other divalent metal ions. At a 17-fold excess Mn(2+), Mg(2+), Ca(2+), and Zn(2+) showed no inhibition of (63)Ni(2+) transport but Co(2+), Cd(2+), and Cu(2+) inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4 degrees C), by protonophores (carbonyl cyanide m-chlorophenylhydrazone, 44%, and 2,4-dinitrophenol, 26%), by sodium azide (25%), and hydroxyl amine (33%). Inhibitors of ATP synthase (N, N'-dicyclohexylcarbodiimide and oligomycin) and incubation of cells in the dark stimulated Ni(2+) transport. (63)Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated (63)Ni(2+) accumulation coincided with the appearance of CODH activity in the culture, suggesting that the (63)Ni(2+) was accumulating in CODH. The cooC, cooT, and cooJ genes are required for the increased (63)Ni(2+) accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated (63)Ni(2+) like cells unexposed to CO. 相似文献
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Recently we discovered a tobacco protein (designated NtCBP4) that modulates heavy-metal tolerance in transgenic plants. Structurally, NtCBP4 is similar to mammalian cyclic-nucleotide-gated non-selective cation channels containing six putative transmembrane domains, a predicted pore region, a conserved cyclic-nucleotide-binding domain, and a high-affinity calmodulin-binding site that coincides with its cyclic-nucleotide-binding domain. Transgenic tobacco expressing the plasma-membrane-localized NtCBP4 exhibit improved tolerance to Ni(2+) and hypersensitivity to Pb(2+), which are associated with a decreased accumulation of Ni(2+) and an enhanced accumulation of Pb(2+) respectively. Transgenic plants expressing a truncated version of NtCBP4, from which regulatory domains had been removed, have a different phenotype. Here we describe our approach to studying the involvement of NtCBP4 in heavy-metal tolerance and to elucidate its physiological role. 相似文献
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目的:基于转酮酶基因缺失菌株MG1655-ΔtktA,研究启动子替换L-组氨酸操纵子前导区及6-磷酸葡萄糖脱氢酶基因zwf、6-磷酸葡萄糖酸脱氢酶基因gnd、PRPP合成酶基因prs的过表达对大肠杆菌产L-组氨酸的影响。方法:通过Red重组系统用T5启动子替换L-组氨酸操纵子前导区;构建gnd和zwf串联表达载体gnd-zwf-pSTV28,prs表达载体prs-pQE30。通过摇瓶发酵,考察上述改造对大肠杆菌积累L-组氨酸的影响。结果:测定结果显示,改造菌株的发酵液中均能实现L-组氨酸积累,平均分别为MG1655-ΔtktA-PT5,60.12 mg/L;MG1655-ΔtktA-PT5(prs-pQE30),66.47mg/L;MG1655-ΔtktA-PT5(zwf-gnd-pSTV28),89.69 mg/L;MG1655-ΔtktA-PT5(prs-pQE30,zwf-gnd-pSTV28),111.56 mg/L。结论:L-组氨酸操纵子前导区的修饰使菌株合成L-组氨酸的能力大大增强,而氧化戊糖磷酸途径的加强和PRPP合成酶活性的提高能够进一步提高产量。 相似文献
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Epithelial sodium channels (ENaC) are regulated by various intracellular and extracellular factors including divalent cations. We studied the inhibitory effect and mechanism of external Ni(2+) on cloned mouse alpha-beta-gamma ENaC expressed in Xenopus oocytes. Ni(2+) reduced amiloride-sensitive Na(+) currents of the wild type mouse ENaC in a dose-dependent manner. The Ni(2+) block was fast and partially reversible at low concentrations and irreversible at high concentrations. ENaC inhibition by Ni(2+) was accompanied by moderate inward rectification at concentrations higher than 0.1 mm. ENaC currents were also blocked by the histidine-reactive reagent diethyl pyrocarbonate. Pretreatment of the oocytes with the reagent reduced Ni(2+) inhibition of the remaining current. Mutations at alphaHis(282) and gammaHis(239) located within the extracellular loops significantly decreased Ni(2+) inhibition of ENaC currents. The mutation alphaH282D or double mutations alphaH282R/gammaH239R eliminated Ni(2+) block. All mutations at gammaHis(239) eliminated Ni(2+)-induced inward current rectification. Ni(2+) block was significantly enhanced by introduction of a histidine at alphaArg(280). Lowering extracellular pH to 5.5 and 4.4 decreased or eliminated Ni(2+) block. Although alphaH282C-beta-gamma channels were partially inhibited by the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), alpha-beta-gamma H239C channels were insensitive to MTSET. From patch clamp studies, Ni(2+) did not affect unitary current but decreased open probability when perfused into the recording pipette. Our results suggest that external Ni(2+) reduces ENaC open probability by binding to a site consisting of alphaHis(282) and gammaHis(239) and that these histidine residues may participate in ENaC gating. 相似文献
6.
The heavy metal nickel is an essential mineral trace nutrient found at low concentrations in most natural soils. However, it may reach toxic levels in certain areas and affect a number of biochemical and physiological processes in plants. Wilting and leaf necrosis have been described as typical visible symptoms of Ni(2+) toxicity. The plasma membrane (PM) of root cells constitutes the first barrier for the entry of heavy metals but also a target of their toxic action. This work studies the relationship between disturbances of membrane functionality and the development of the typical symptoms of Ni(2+) toxicity. Rice plants (Oryza sativa L. cv. Bahia) grown in nutrient medium containing 0.5mM Ni(2+) showed a significant decrease in water content as a consequence of the stress. Addition of Ni(2+) to the solution bathing the roots induced a concentration-dependent PM depolarization but the activity of the PM-H(+)-ATPase was not inhibited by the presence of Ni(2+) and the initial resting potential recovered in less than 1h. In the short term (hours), membrane permeability of root cells was not significantly affected by Ni(2+) treatments. However, in the long term (days) a drastic loss of K(+) was measured in roots and shoots, which should be responsible for the changes in the water content measured, since stomatal conductance and the transpiration rate remained unaffected by Ni(2+) treatment. The effects induced by Ni(2+) were not permanent and could be reverted, at least in part, by transferring the plants to a medium without Ni(2+). 相似文献
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Takahashi K Akaishi E Abe Y Ishikawa R Tanaka S Hosaka K Kubohara Y 《Biochemical and biophysical research communications》2003,307(1):64-68
Calcineurin (CN) is a Ca(2+)/calmodulin (CaM)-dependent protein serine/threonine phosphatase that contains Zn(2+) in its catalytic domain and can be stimulated by divalent ions such as Mn(2+) and Ni(2+). In this study, the role of exogenous Zn(2+) in the regulation of CN activity and its relevance to the role of Ni(2+) was investigated. Zn(2+) at a concentration range of 10nM-10 micro M inhibited Ni(2+)-stimulated CN-activity in vitro in a dose-dependent manner and approximately 50% inhibition was attained with 0.25 micro M Zn(2+). Kinetic analysis showed that Zn(2+) inhibited the activity of CN by competing with Ni(2+). Interaction of CN and CaM was not inhibited with Zn(2+) at 10 micro M. Zn(2+) never affected the activity of cAMP phosphodiesterase 1 or myosin light-chain kinase (CaM-dependent enzymes) and rather activated alkaline phosphatase. The present results indicate that Zn(2+) should be a potent inhibitor for CN activity although this ion is essential for CN. 相似文献
8.
Although induction of cell apoptosis is known to be involved in the cytotoxicity of Ni(2+), little research has been aimed at the mechanism of Ni(2+)-induced apoptosis. Recent studies showed that Ni(2+) induces histone hypoacetylation in different cell lines. Since histone hypoacetylation plays important roles in the control of cell cycle progress and apoptosis, we hypothesized that histone hypoacetylation may be an unrevealed pathway in Ni(2+)-induced apoptosis. To address this, effects of Ni(2+) on cell apoptosis, bcl- 2 gene expression and histone acetylation were examined in human hepatoma Hep3B cells. We found that Ni(2+) treatment resulted in cell proliferation arrest, the appearance of detached cells, condensed chromatin, apoptotic bodies and specific DNA fragmentation, indicating the occurrence of cell apoptosis. At the same time, Ni(2+) induced a significant decrease in bcl- 2 expression and histone acetylation; the decrease of histone H4 acetylation in nucleosomes associated with the bcl- 2 promoter region was also proven by a chromatin immunoprecipitation assay, indicating the involvement of histone hypoacetylation in Ni(2+)-induced bcl- 2 down-regulation. Further studies showed that increasing histone acetylation by either 100 nM of trichostatin A or over-expressing histone acetyltranferase p300 in Hep3B cells obviously attenuated the bcl- 2 down-regulation and cell apoptosis caused by Ni(2+). Considering the importance of bcl- 2 in determining cell survival and apoptosis, the data presented here suggest that histone hypoacetylation may represent one unrevealed pathway in Ni(2+)-induced cell apoptosis, where bcl- 2 is one of its targets. 相似文献
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Ha EJ Kim YJ An SS Kim YR Lee JO Lee SG Paik HJ 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2008,876(1):8-12
In this study, a new matrix for immobilized metal affinity chromatography (IMAC) using poly(2-acetamidoacrylic acid) (PAAA) hydrogels complexed with Ni(2+) was developed for the purification of the recombinant histidine-tagged green fluorescence protein (His6-GFP). The Ni(2+)-complexed PAAA hydrogel was prepared by polymerizing 2-acetamidoacrylic acid (AAA) and 2,2'-[(1,4-dioxo-1,4-butanediyl)diamino] bis(2-propenoic acid) (DBDBPA) with potassium persulfate in DMSO, followed by Ni(2+) complexation. Confocal laser scanning microscopy was used to determine the binding of His6-GFP to the Ni(2+)-PAAA hydrogel in three-dimensional space. Photoluminescence spectroscopy revealed an 81% binding efficiency of His6-GFP to the Ni(2+)-PAAA hydrogel yielded with a recovery of 59%. The specificity of His6-GFP binding to Ni(2+)-PAAA hydrogel was compared with that of the PAAA hydrogel without Ni(2+). His6-GFP was purified directly from the cell lysate with Ni(2+)-PAAA hydrogel matrix but the PAAA hydrogel without Ni(2+) had no effect. The major advantage of the Ni(2+)-PAAA hydrogel system over current methods, such as Ni-nitrilotriacetic acid (NTA) agarose beads, was the simple and low-cost procedure for preparing the matrix. 相似文献
11.
The effect of nickel on secretory systems. Studies on the release of amylase, insulin and growth hormone 总被引:5,自引:3,他引:2
Robert L. Dormer Alan L. Kerbey Margaret McPherson Susan Manley Stephen J. H. Ashcroft J. George Schofield Philip J. Randle 《The Biochemical journal》1974,140(2):135-142
The effects of Ni(2+) on the release of amylase from rat parotids, insulin from mouse pancreatic islets and growth hormone from bovine pituitary slices were investigated. In all these secretory systems, Ni(2+) was shown to inhibit release evoked by a variety of stimuli both physiological and pharmacological. Measurements of rates of substrate oxidation and tissue concentrations of ATP and 3':5'-cyclic AMP suggest that this inhibitory action of Ni(2+) does not arise through an effect on energy metabolism or cyclic AMP metabolism. It is concluded that although some effects of Ni(2+) may involve antagonism between Ni(2+) and Ca(2+) in stimulus-secretion coupling, others appear to be independent of Ca(2+). It is suggested that Ni(2+) may block exocytosis by interfering with either secretory-granule migration or membrane fusion and microvillus formation. The possible mode of action of Ni(2+) and its potential use as a tool in the study of exocytosis are discussed. 相似文献
12.
Ni(2+) inhibits current through calcium channels, in part by blocking the pore, but Ni(2+) may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of Ni(2+) on Ca(V)3.1 is affected by permeant ions. We find two components to block by Ni(2+), a rapid block with little voltage dependence, and a slow block most visible as accelerated tail currents. Rapid block is weaker for outward vs. inward currents (apparent K(d) = 3 vs. 1 mM Ni(2+), with 2 mM Ca(2+) or Ba(2+)) and is reduced at high permeant ion concentration (110 vs. 2 mM Ca(2+) or Ba(2+)). Slow block depends both on the concentration and on the identity of the permeant ion (Ca(2+) vs. Ba(2+) vs. Na(+)). Slow block is 2-3x faster in Ba(2+) than in Ca(2+) (2 or 110 mM), and is approximately 10x faster with 2 vs. 110 mM Ca(2+) or Ba(2+). Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations ( approximately 3 muM Ca(2+)). We conclude that both fast and slow block of Ca(V)3.1 by Ni(2+) are most consistent with occlusion of the pore. The exit rate of Ni(2+) for slow block is reduced at high Ni(2+) concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni(2+), at a site located deeper within the pore. In contrast to the complex pore block observed for Ca(V)3.1, inhibition of Ca(V)3.2 by Ni(2+) was essentially independent of voltage, and was similar in 2 mM Ca(2+) vs. Ba(2+), consistent with inhibition by a different mechanism, at a site outside the pore. 相似文献
13.
Conformational changes induced by the binding of nickel to horseradish peroxidase C (HRPC) were studied by electronic absorption spectroscopy, fluorescence spectroscopy and circular dichroism spectroscopy. Incubation of HRPC with various concentrations of Ni(2+) for 5 minutes resulted in changes in the enzyme absorption spectrum, including variations in the intensities of the Soret, beta and charge transfer (CT1) bands absorption, shift in the Soret, beta and CT1 bands maxima and absorption increase at 275 nm. Increases in the enzyme's intrinsic fluorescence as determined by fluorescence spectroscopy, as well as changes in the alpha-helical content, as determined by circular dichroism spectroscopy, were also found. Correlatively, alterations of the enzymatic activity by Ni(2+) were studied by following the H(2)O(2)-mediated oxidation of o-dianisidine and 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) by HRPC. With both reducing substrates, it was found that in the presence of sufficient amount of enzyme, 1-10 mM nickel would enhance the enzymatic activity, while higher Ni(2+) concentrations (20-50 mM) would inhibit it. The enzyme was completely inhibited after 5 minutes incubation in 50 mM Ni(2+). Prolonged incubation would induce complete inhibition at lower Ni(2+) concentrations. Spectrophotometry investigations also showed that inhibitory concentrations of Ni(2+) altered compounds I and II formation, compound II being the first affected. Based on spectrophotometry, fluorescence and circular dichroism spectroscopy, and data on compounds I and II formation, a scheme is suggested for HRPC conformational changes in different Ni(2+) concentrations. HRPC was found to have four potential attachment sites for Ni(2+) which were sequentially occupied in a dose- and time-dependent manner by the metallic ion. 相似文献
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Influence of Ni(2+) concentration on biohydrogen production 总被引:2,自引:0,他引:2
In this paper, the effect of Ni(2+) concentration ranging from 0 to 50mg/L on fermentative hydrogen production by mixed cultures was investigated in batch test. The results showed that at 35 degrees C and initial pH 7.0, Ni(2+) was able to enhance the hydrogen production rate with increasing Ni(2+) concentration from 0 to 0.2mg/L, and enhance the hydrogen production potential and hydrogen yield with increasing Ni(2+) concentration from 0 to 0.1mg/L. The maximum hydrogen production potential of 288.6mL and the maximum hydrogen yield of 296.1mL/g glucose were obtained at the Ni(2+) concentration of 0.1mg/L. In all tests, the major soluble metabolites produced by mixed cultures were ethanol, acetic acid and butyric acid, without propionic acid. Ni(2+) had little effect on the substrate degradation efficiency with increasing concentration from 0 to 50mg/L. Ni(2+) was able to enhance the biomass production yield with increasing Ni(2+) concentration from 0 to 0.1mg/L. The maximum biomass production yield of 232.5mg/g glucose was obtained at the Ni(2+) concentration of 0.1mg/L. In all tests, the final pH after fermentative hydrogen production was lower than the initial pH. 相似文献
16.
Manini P Napolitano A Camera E Caserta T Picardo M Palumbo A d'Ischia M 《Biochimica et biophysica acta》2003,1621(1):9-16
Ni(2+), a toxic, carcinogenic and allergenic agent, affected both the kinetic and chemical courses of the Fe(2+)-induced oxidation of arachidonic acid (AA) in 0.05 M phosphate buffer (pH 7.4) and at 37 degrees C. At 10 microM concentration, Ni(2+) decreased the rate of oxidation of peroxide-free AA (200 microM) promoted by 50 microM Fe(2+), as determined by measurement of thiobarbituric acid reactive species (TBARS) and 1H NMR analysis. However, in the presence of low levels of peroxides (e.g. 2%), Ni(2+) exerted a significant stimulatory effect on Fe(2+)-induced AA oxidation and TBARS formation. 1H NMR analysis showed that Ni(2+) (10 microM) enhanced formation of genotoxic alkenals including 4-hydroxy-2-nonenal (4-HNE, GC/MS evidence) by Fe(2+)-promoted degradation of both AA and 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) methyl esters. The observed stimulatory effects of Ni(2+) on peroxide breakdown and cytotoxic aldehyde formation provide an attractive explanation to the enhanced sensitization capacity of nickel in inflammatory states compared to normal states. 相似文献
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NreB from Achromobacter xylosoxidans 31A Is a nickel-induced transporter conferring nickel resistance 总被引:4,自引:0,他引:4
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There are two distinct nickel resistance loci on plasmid pTOM9 from Achromobacter xylosoxidans 31A, ncc and nre. Expression of the nreB gene was specifically induced by nickel and conferred nickel resistance on both A. xylosoxidans 31A and Escherichia coli. E. coli cells expressing nreB showed reduced accumulation of Ni(2+), suggesting that NreB mediated nickel efflux. The histidine-rich C-terminal region of NreB was not essential but contributed to maximal Ni(2+) resistance. 相似文献
18.
Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions 总被引:1,自引:0,他引:1
Kuznetsova IA Orlovskaya IA Buneva VN Nevinsky GA 《Biochimica et biophysica acta》2007,1774(7):884-896
We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me(2+)) ions, with the level of activity decreasing in the order Mn(2+)> or =Mg(2+)>Ca(2+)> or =Cu(2+)>Co(2+)> or =Ni(2+)> or =Zn(2+), whereas Fe(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me(2+) ions in the 0.1-12 mM range, depending on the particular metal ion. In the presence of all Me(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu(2+), Zn(2+), or Ca(2+) inhibited the Mg(2+)-dependent hydrolysis of scDNA, while Ni(2+), Co(2+), and Mn(2+) activated this reaction. The Mn(2+)-dependent hydrolysis of scDNA was activated by Ca(2+), Ni(2+), Co(2+), and Mg(2+) ions but was inhibited by Cu(2+) and Zn(2+). After addition of the second metal ion, only in the case of Mg(2+) and Ca(2+) or Mn(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0-100%) in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed. 相似文献
19.
Ni(II) and Zn(II) M-DNA formation and denaturation of double-stranded DNA (dsDNA) by Cd(2+) were monitored by surface plasmon resonance (SPR). When exposed to immobilized 30 bp 50% GC dsDNA, Zn(2+) and Ni(2+) were found to give signals indicative of a conformational change at pH 8.5 but not 7.5, while Mg(2+) and Ca(2+) caused small changes at both pHs. The concentrations that gave 50% of the maximum responses were 0.06 and 0.50 mM for Zn(2+) and Ni(2+), respectively. At pH 8.5, Cd(2+) denatured over 40% of the dsDNA, while other metals denatured less than 5% of the DNA. Smaller pH-dependent signals were induced by Zn(2+), Ni(2+) or Cd(2+) with 50% GC single-stranded DNA (ssDNA), and with a homopolymer of d(T)30. Homopolymers d(A)30 and d(C)30 showed small signals that were largely independent of pH in the presence of Zn(2+) or Ni(2+). 相似文献
20.
Streptomyces coelicolor mutant RF-59, isolated as a revertant of a histidine auxotroph after mutagenic treatment with N-methylN'-nitro-N-nitrosoguanidine, was found to accumulate L-histidine. The mutant was sensitive to 2-thiazo-lealanine and L-2,4-diaminobutyric acid and partially sensitive to alpha-methylhistidine but resistant to 1,2,4-triazolealanine, indicating that repression of the histidine operon was modified in the mutant. Culture conditions were investigated, and optimal media for L-histidine production were developed, resulting in L-histidine accumulation of 2.1 to 3.5 g/liter. 相似文献