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1.
We constructed and characterized a Xanthomonas campestris pv. phaseoli oxyR mutant. The mutant was hypersensitive to H 2O 2 and menadione killing and had reduced aerobic plating efficiency. The oxidants’ induction of the catalase and ahpC genes was also abolished in the mutant. Analysis of the adaptive responses showed that hydrogen peroxide-induced protection against hydrogen peroxide was lost, while menadione-induced protection against hydrogen peroxide was retained in the oxyR mutant. These results show that X. campestris pv. phaseoli oxyR is essential to peroxide adaptation and revealed the existence of a novel superoxide-inducible peroxide protection system that is independent of OxyR. 相似文献
2.
Generation of reactive oxygen species (ROS) constitutes an important first reaction under many stress conditions in plants.
We demonstrate that Nicotiana tabacum L. cv. Bright Yellow 2 (TBY-2) cells in suspension cultures, generate superoxide radical and hydrogen peroxide upon treatment
with cadmium and zinc. Addition of catalase and N, N-diethyldithiocarbamate (DDC) decreased the level of H 2O 2, whereas superoxide dismutase (SOD) induced a slight increase of the H 2O 2 production. The effects of catalase, DDC and SOD on the heavy metal-induced ROS production indicate that it occurs outside
of the cells, and that at least part of the hydrogen peroxide is produced by dismutation of the superoxide radical (O 2
·−). The effect of pretreatment of the cell cultures with commonly used mammalian NADPH oxidase inhibitors was also tested.
Strong inhibitions of cadmium and zinc-mediated ROS production were obtained with the flavoprotein inhibitors—diphenylene
iodonium (DPI) and quinacrine and with an inhibitor of b-type cytochromes—imidazol. Membrane permeable- N-ethyl maleimide (NEM) and iodoacetate, and membrane non-permeable thiol reagents—para-chloromercuribenzoic acid (pCMBS) also
inhibited the ROS production. These results suggested that the enzyme responsible for cadmium and zinc-induced ROS production
in tobacco cells contains a flavocytochrome. They also show the importance of intra- and extracellular thiol groups in the
observed stress reaction. The induction of ROS production with heavy metals showed properties comparable to the elicitor-induced
oxidative burst in other plant cells. 相似文献
3.
All of the δ, μ, and κ opioid receptors have a free thiol group of the Cys residue in the ligand-binding site, although its functional role is not yet known. In order to examine whether or not a similar Cys is also present in the ORL1 nociceptin receptor, we attempted to identify it by affinity labeling using a specific antagonist peptide. We first treated ORL1-expressing COS-7 cell membrane preparations with the thiol-alkylation reagent N-ethylmaleimide (NEM) to perform a binding assay using [ 3H]nociceptin as a tracer and nociceptin, an ORL1 agonist, or Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH 2, a nociceptin/ORL1 antagonist, as a competitor. It was suggested that ORL1 has a free Cys in its ligand-binding site, since the NEM treatment reduced the population of ligand-binding sites. This was further confirmed by affinity labeling using Cys(Npys)-Arg-Tyr-Tyr-Arg-Ile-Lys-NH 2 with the SNpys group that can react with a free thiol group, resulting in the formation of a disulfide bond. This affinity labeling was approximately 23 times more specific than NEM alkylation. The results revealed that the ORL1 nociceptin receptor does contain a free Cys residue in the ligand-binding site. 相似文献
6.
Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill both Gram‐positive and Gram‐negative bacteria through simultaneous induction of oxidative, thiol and metal stress responses in bacteria. However, metabolic pathways through which PGRPs induce these bactericidal stress responses are unknown. We screened Keio collection of Escherichia coli deletion mutants and revealed that deleting genes for respiratory chain flavoproteins or for tricarboxylic acid (TCA) cycle resulted in increased resistance of E. coli to PGRP killing. PGRP‐induced killing depended on the production of hydrogen peroxide, which required increased supply of NADH for respiratory chain oxidoreductases from central carbon catabolism (glycolysis and TCA cycle), and was controlled by cAMP‐Crp. Bactericidal PGRP induced a rapid decrease in respiration, which suggested that the main source of increased production of hydrogen peroxide was a block in respiratory chain and diversion of electrons from NADH oxidoreductases to oxygen. CpxRA two‐component system was a negative regulator of PGRP‐induced oxidative stress. By contrast, PGRP‐induced thiol stress (depletion of thiols) and metal stress (increase in intracellular free Zn 2+ through influx of extracellular Zn 2+) were mostly independent of oxidative stress. Thus, manipulating pathways that induce oxidative, thiol and metal stress in bacteria could be a useful strategy to design new approaches to antibacterial therapy. 相似文献
7.
In Escherichia coli hosts, hydrogen peroxide is one of the factors that may cause induction of λ prophage. Here, we demonstrate that H 2O 2-mediated λ prophage induction is significantly enhanced in the oxyR mutant host. The mRNA levels for cI gene expression were increased in a λ lysogen in the presence of H 2O 2. On the other hand, stimulation of the p
M promoter by cI857 overproduced from a multicopy plasmid was decreased in the Δ oxyR mutant in the presence of H 2O 2 but not under normal growth conditions. The purified OxyR protein did bind specifically to the p
M promoter region. This binding impaired efficiency of interaction of the cI protein with the OR3 site, while stimulating such
a binding to OR2 and OR1 sites, in the regulatory region of the p
M promoter. We propose that changes in cI gene expression, perhaps in combination with moderately induced SOS response, may be responsible for enhanced λ prophage
induction by hydrogen peroxide in the oxyR mutant. Therefore, OxyR seems to be a factor stimulating λ prophage maintenance under conditions of oxidative stress. This
proposal is discussed in the light of efficiency of induction of lambdoid prophages bearing genes coding for Shiga toxins. 相似文献
8.
Chemical oxidation or reduction of lymphocyte cell surface thiol or disulfide groups, respectively, has been shown to alter the proliferative activity of murine T cells. S-2-(3-aminopropylamino)ethylphosphothioic acid, a compound containing no free thiol group until it is intracellularly dephosphorylated, did not enhance Con A-induced proliferation which suggested that thiols did not mediate proliferative enhancement via an intracellular mechanism. Glutathione, an impermeant thiol, enhanced T-cell proliferation 68% as effectively as 2-mercaptoethanol (2-ME), which suggested that the thiol-sensitive site was at the cell surface. A battery of structural analogs to 2-ME was employed to elucidate the chemical requirements for the biological activity of the thiols. The necessity for a hydrogen-binding moiety on the thiol reagent was determined by the use of non-hydrogen-binding analogs and by competitive inhibition of the thiol-enhancing activity of 2-ME by non-thiol-containing hydrogen-binding analogs. Pretreatment of cells with the copper:phenanthroline complex (CuP), an impermeant oxidant of thiol groups, reduced the Con A-induced response >79%; however, the presence of 2-ME in culture completely reversed the inhibitory effect of CuP pretreatment. Oxidation of T cells by high oxygen tension (17% O 2) also ablated the Con A response but did not alter the response to Con A + 2-ME. Protection from oxidative inhibition also was afforded T cells by sequential reduction and blockage of cell surface thiol groups. Finally, a model which correlates the chemical study of cell surface residues with T-lymphocyte responsiveness is presented. 相似文献
9.
A novel OxyR (DR0615) with one conserved cysteine that senses hydrogen peroxide in Deinococcus radiodurans had been identified in our previous work. Comparative genomics revealed that D. radiodurans possesses another OxyR homolog, OxyR 2 (DRA0336). In this study, we constructed the deletion mutant of oxyR
2
and the double mutant of both the OxyR homologs to investigate the role of OxyR in response to oxidative stress in D. Radiodurans. Deletion of oxyR
2
resulted in an obviously increased sensitivity to hydrogen peroxide, and the double mutant for oxyR and oxyR
2 was significantly more sensitive than any of the two single mutants. The total catalase activity of the double mutant was
lower than that of any of the single mutants, and reactive oxygen species (ROS) accumulated to a greater extent. DNA microarray
analysis further suggested that oxyR
2
was involved in antioxidation mechanisms. Site-direct mutagenesis and complementation analysis revealed that C 228 in OxyR 2 was essential. This is the first report of the presence of two OxyR in one organism. These results suggest that D. radiodurans OxyR and OxyR 2 function together to protect the cell against oxidative stress. 相似文献
10.
Nitrate uptake in right-side out plasma membrane vesicles isolated from cucumber roots was characterized. Nitrate uptake into
vesicles was driven by an artificially imposed pH gradient. The uptake was strongly inhibited by phenylglyoxal, an arginyl
residue modificator. Only a slight repression of NO
3
−
transport in vesicles was observed in the presence of NEM, a thiol group reagent. pCMBS, an other thiol reagent and DEPC, an effector of histidine residue, had no effect on the nitrate transport in plasma
membranes. ATP-driven proton transport in vesicles was not significantly affected in the presence of both, phenylglyoxal and
DEPC, whereas pCMBS and NEM abolished it almost completely. The possible role of the particular amino acids residues in the active nitrate
transport is discussed.
NO
3
−
uptake into vesicles isolated from both, nitrate-induced and nitrate-depleted plant material was higher than that observed
in the vesicles obtained from uninduced plants. Thus, isolated vesicles reflect the well-known in vivo response of intact plants on the exogenous nitrogen regime. 相似文献
11.
An approach to decontamination of biological endospores is discussed. Specifically, the performance of an aqueous modified Fenton reagent is examined. A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions. The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions. Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu 2+) and then subsequent conversion to hydroxyl radicals. Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii. A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature. This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O 2 is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action. 相似文献
12.
Among all polyphenols tested (tannic acid and flavonoids belonging to different subclasses) only tannin and quercetin significantly enhanced resistance of Escherichia coli to peroxide stress. Pretreatment of the cells with quercetin and tannin resulted in a decrease in the growth arrest duration under moderate H 2O 2 concentration (2 mM) and an increase in survival under high (10 mM) doses. The shorter growth recovery period in pretreated cells was connected with more rapid H 2O 2 elimination because of induced activity of scavenging enzymes. This effect was absent in the Δ oxyR mutant, which was unable to induce genes responding to peroxide stress. The data obtained suggest that the observed protection was a result of two overlapping effects: induction of OxyR regulon by low concentrations of H 2O 2, accumulated during extracellular autoxidation of quercetin and tannin, and protection of synthesis of OxyR-regulated antioxidant enzymes during H 2O 2 stress because of intracellular binding of iron by quercetin and tannin and suppressing Fenton chemistry. 相似文献
13.
Pseudomonas chlororaphis GP72 is an important plant growth-promoting rhizobacteria (PGPR) with a wide-spectrum antibiotic activity toward several soil-borne pathogens. The adaption of this strain to different environmental oxidative stress and redox phenazine pigment by the predicted regulator OxyR were investigated. The deletion of oxyR led to a significant reduction of the viability, production of three phenazine derivatives and resistance to hydrogen peroxide and paraquat on the KB agar plates. However, the mutant ΔoxyR grew better with shorter delay. In addition, the mutant ΔoxyR showed an increased resistance to hydrogen peroxide, which occurred at the concentration varying from 1.0 mM to 5.0 mM in the KB broth, as compared with the wild type. In addition, the biofilm formation ability was obviously enhanced and influenced by the different oxidants in the mutant. Quantitative RT-PCR experiments indicated that the expression of katG, ahpC, ahpD and phzE were increased in the oxyR mutant background in response to hydrogen peroxide. katG was mainly responsible for the enhanced resistance to hydrogen peroxide. The loss of oxyR is suggested to benefit the hydrogen peroxide inducible gene expression. Thus, OxyR is an important global regulator that regulates multiple pathways to enhance the survival of P. chlororaphis GP72 exposed to different oxidative stresses. 相似文献
14.
Thiol redox state (TRS) refers to the balance between reduced thiols and their corresponding disulfides and is mainly reflected by the ratio of reduced and oxidized glutathione (GSH/GSSG). A decrease in GSH/GSSG, which reflects a state of thiol oxidative stress, as well as thiol modifications such as S-glutathionylation, has been shown to have important implications in a variety of cardiovascular diseases. Therefore, research models for inducing thiol oxidative stress are important tools for studying the pathophysiology of these disease states as well as examining the impact of pharmacological interventions on thiol pathways. The purpose of this study was to evaluate the use of a dithiocarbamate derivative, 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), as a pharmacological model of thiol oxidative stress by examining the extent of thiol modifications induced in H9c2 rat cardiomyocytes and its impact on cellular functions. The extent of thiol oxidative stress produced by 2-AAPA was also compared to other models of oxidative stress including hydrogen peroxide (H 2O 2), diamide, buthionine sulfoximine, and N,N׳-bis(2-chloroethyl)- N-nitroso-urea. Results indicated that 2-AAPA effectively inhibited glutathione reductase and thioredoxin reductase activities and decreased the GSH/GSSG ratio by causing a significant accumulation of GSSG. 2-AAPA also increased the formation of protein disulfides as well as S-glutathionylation. The alteration in TRS led to a loss of mitochondrial membrane potential, release of cytochrome c, and increase in reactive oxygen species production. Compared to other models, 2-AAPA is more potent at creating a state of thiol oxidative stress with lower cytotoxicity, higher specificity, and more pharmacological relevance, and could be utilized as a research tool to study TRS-related normal and abnormal biochemical processes in cardiovascular diseases. 相似文献
15.
We have studied the amino-acid residues involved in the catalytic activity of two distinct brain sialyltransferases acting on fetuin and asialofetuin. These two enzymes were strongly inhibited by N-bromosuccinimide, a specific blocking reagent for tryptophan residues. This result suggests the involvement of such residues in the catalytic process of the two sialytransferases. Furthermore, chemical modifications by various sulfhydryl reagents led to a strong inhibition of the fetuin sialyltransferase while the asialofetuin sialyltransferase was only slightly inhibited. For a more thorough understanding of the thiol inactivation mechanism of the fetuin sialyltransferase, we studied in more detail the reactivity of this enzyme with NEM ( N-ethylmaleimide), an irreversible reagent. The time-dependent inactivation followed first-order kinetics and these kinetic data afforded presumptive evidence for the binding of 1 mol NEM per mol of enzyme. Only CMP-NeuAc protected the enzyme against NEM inactivation effectively. MnCl 2 did not enhance the protective effect of CMP-NeuAc. The modifications of the fetuin sialyltransferase kinetic parameters by NEM showed a competitive mechanism between NEM and CMP-NeuAc. The results suggest the involvement of a sulfhydryl residue in or near the nucleotide-sugar binding may induce a change in conformation of the protein, leading to a decreased accessibility of this thiol group located near the nucleotide-sugar binding site). This SH group, is essential to the enzyme activity, which is not the case for the asialofetuin sialyltransferase.Abbreviations
p-CMB
p-chloromercuribenzoic acid
- CPDS
6,6-dithiodinicotinic acid carboxypyridine disulfide
- DTNB
5,5-dithiobis-(2-nitrobenzoic acid)
- NEM
N-ethylmaleimide
- DTT
dithiothreitol
- Mes
2-( N-morpholino)ethane sulfonic acid
- NeuAc
N-acetylneuraminic acid 相似文献
16.
A moderate reduction in the non-protein thiol content of V79 379A Chinese hamster cells, obtained by pretreatment with buthionine sulphoximine (BSO), diethyl maleate (DEM) or N-ethyl maleimide (NEM), increase both the absolute radiosensitivity of the cells in hypoxia and the radiosensitizing effect of adding oxygen 7 ms after irradiation. Combined pretreatment of cells with BSO and NEM removes most of the non-protein thiol and some of the protein thiol; such treatment further increases the radiosensitivity of hypoxic cells but there is no further effect of adding oxygen 7 ms after irradiation. Addition of 2-mercaptoethanol to cells 7 ms after irradiation gives protection factors that increase with increasing severity of thiol depletion. Substantial radioprotection can still be observed when 2-mercaptoethanol is added 70 ms after irradiation of cells pretreated with BSO and NEM; there is no effect of adding 2-mercaptoethanol to such cells 50s after irradiation. These observations support the repair-fixation model of radiation damage and suggest that, in addition to the established role of non-protein thiol in chemical repair of radiation damage, other endogenous reducing agents such as protein thiol may be important in determining cellular radiosensitivity. A relatively long-lived thiol-modifiable component of radiation damage has been observed within hypoxic thiol-depleted cells. 相似文献
17.
Low doses of
-cysteine (CYS), cysteinyl-glycine (CYSGLY) and reduced glutathione (GSH) activated by γ-glutamyl transpeptidase (GGT) were mutagenic in strain IC203 ( oxyR), whereas higher doses were required to observe a weak mutagenicity in the oxyR+ strain WP2 uvrA/pKM101 (denoted IC188). This indicates that thiol mutagenesis is suppressed by OxyR-regulated antioxidant defenses and confirms its oxidative character. The mutagenesis by low doses of CYS, CYSGLY and GSH+GGT detected in IC203 was abolished by rat liver S9, through the activity of catalase, as well as by the metal chelator diethyldithiocarbamate (DETC), supporting the dependence of this mutagenesis on H 2O 2 production, probably in thiol autoxidation reactions in which transition metals are involved. Surprisingly, low DETC concentrations greatly potentiate the mutagenicity of low CYS doses. Mutagenesis by high doses of CYS and CYSGLY occurred in both IC203 and IC188 in the presence of liver S9, and was resistant to inhibition by catalase, although it was prevented by DETC. Mutagenesis by GSH activated by rat kidney S9, rich in GGT, was detected in IC203 and IC188 only at high doses since catalase and glutathione peroxidase, both present in kidney S9, might inhibit its induction by low GSH doses. In the presence of liver S9, almost deficient in GGT, GSH was not mutagenic. The mutagenicity of a high GSH dose occurring in the presence either of GGT plus liver S9 or of kidney S9 was weakly prevented by DETC. 相似文献
18.
It was found that modification of thiol (SH-) groups of membrane proteins by Ellman’s reagent (5,5′-dithiol-bis-(2-nitrobenzoic) acid) results in inhibition of proton efflux and K + influx in anaerobically grown (pH 7.5) wild-type strains of Escherichia coli and causes disturbances in K +-dependent, N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity and molecular hydrogen production. No such effects were observed after substitution of the cysteine residue in the b-subunit of F 0 of proton F 0F 1-ATPase for alanine. Moreover, the redox potential (RP) decreased as a result of H 2 release during glucose fermentation and formate utilization was partly restored in the presence of Ellman’s reagent. Similar changes were established when another specific SH-reagent, succinimidyl-6(β-maleimidopropionamido)hexanoate, was used. Another thiol reagent, N-ethylmaleimide, did not exert such effects despite its inhibitory action on ion transport and ATPase activity. The data obtained provide conclusive evidence in favor of essential role of thiol groups and the cysteine residue in the b-subunit of F 0 of F 0F 1-ATPase in proton-potassium exchange and H 2 production in E. coli cells. The results also point to a possible involvement of SH-groups in the TrkA system of K + uptake and an involvement of hydrogenases 3 or 4 in the interactions of these integral proteins with each other. 相似文献
19.
Influx isotherms were obtained for 86Rb + uptake into 2-cm corn ( Zea mays [A632 × (C3640 × Oh43)] root segments for both low- (0.2 millimolar CaSO 4) and high-salt (0.2 millimolar CaSO 4 + 5 millimolar KCl) grown roots. Unlike the discontinuous curves usually presented for K + influx, our isotherms were smooth, nonsaturating curves that approached linearity at K + (Rb +) concentrations above 1 millimolar. The kinetics for K + transport could be resolved into saturable and linear components. The saturable components yielded Km values of 16 and 86 micromolar for low- and high-salt roots, respectively, while Vmax values were 5.62 and 1.85 moles per gram fresh weight per hour. Results of experiments with the penetrating sulfhydryl reagent, N-ethyl maleimide (NEM), and the impermeant reagent, p-chloromercuribenzene sulfonic acid (PCMBS) indicated that the saturable and linear components were independent mechanisms of K + transport. Short-term NEM exposures (30 seconds to 5 minutes) selectively inhibited the saturable system, but had little effect on the linear component. Increasing NEM exposures resulted in further inhibition and subsequent abolition of the saturable component; the linear component exhibited limited NEM sensitivity. PCMBS elicited the same general inhibitory trends, although it was less effective as a saturable component inhibitor. The effects of NEM and PCMBS on K+ efflux were also studied. Short NEM exposures had no effect on cytoplasmic efflux, while inhibiting vacuolar efflux significantly. From these data, it is unclear at which site(s) NEM is acting. A more complex response was obtained with PCMBS, where a monophasic efflux curve was observed. Analysis indicated that the vacuolar efflux was stimulated, while the cytoplasmic component was abolished. The nature of the linear component is discussed, and it is proposed that the mechanism may be more complex than simple facilitated diffusion. 相似文献
20.
Vibrio parahaemolyticus is a common marine food-borne enteropathogen. In this study, we examined the antioxidative activity, growth, biofilm formation, and cell mobility of an oxyR deletion mutant and its genetically complementary strain of V. parahaemolyticus. oxyR is the regulator of catalase and ahpC genes. Protection against extrinsic H 2O 2 and against the organic peroxides cumene hydroperoxide and tert-butyl hydroperoxide was weaker in the deletion mutant than in its parent strain. Expression of the major functional antioxidative genes, ahpC1 and VPA1418, was markedly decreased in the oxyR mutant. Growth of this mutant on agar medium was significantly inhibited by autoclaved 0.25% glucose and by 0.25% dipotassium hydrogen phosphate, 0.5% monosaccharides (glucose, galactose, xylose, and arabinose), or 114.8 mM phosphates. The inhibition of the growth of this oxyR mutant by extrinsic peroxides, autoclaved sugars, and phosphates was eliminated by the complementary oxyR gene or by the addition of catalase to the autoclaved medium, while no inhibition of growth was observed when filter-sterilized sugars were used. The formation of biofilm and swimming mobility were significantly inhibited in the oxyR mutant relative to that in the wild-type strain. This investigation demonstrates the antioxidative function of oxyR in V. parahaemolyticus and its possible roles in biofilm formation, cell mobility, and the protection of growth in heated rich medium. 相似文献
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