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PubMed2006

Thionein can serve as a reducing agent for the methionine sulfoxide reductases.

It has been generally accepted, primarily from studies on methionine sulfoxide reductase (Msr) A, that the biological reducing agent for the members of the Msr family is reduced thioredoxin (Trx), although high levels of DTT can be used as the reductant in vitro. Preliminary experiments using both human recombinant MsrB2 (hMsrB2) and MsrB3 (hMsrB3) showed that although DTT can function in vitro as the reducing agent, Trx works very poorly, prompting a more careful comparison of the ability of DTT and Trx to function as reducing agents with the various members of the Msr family. Escherichia coli MsrA and MsrB and bovine MsrA efficiently use either Trx or DTT as reducing agents. In contrast, hMsrB2 and hMsrB3 show <10% of the activity with Trx as compared with DTT, raising the possibility that, in animal cells, Trx may not be the direct hydrogen donor or that there may be a Trx-independent reducing system required for MsrB2 and MsrB3 activity. A heat-stable protein has been detected in bovine liver that, in the presence of EDTA, can support the Msr reaction in the absence of either Trx or DTT. This protein has been identified as a zinc-containing metallothionein (Zn-MT). The results indicate that thionein (T), which is formed when the zinc is removed from Zn-MT, can function as a reducing system for the Msr proteins because of its high content of cysteine residues and that Trx can reduce oxidized T.

Animals↗
PubMed1994

Thiol reducing agents modulate induced apoptosis in porcine endothelial cells.

When cultured porcine endothelial cells are exposed first to endotoxin (lipopolysaccharide (LPS)) followed by standard inducers of the heat shock response in vitro (heat or sodium arsenite), these cells aberrantly execute programmed cell death. This cell death is dependent upon two distinct events: the LPS-priming step and the heat shock-induced activation step. Prior work demonstrated that the LPS-priming step could be blocked by the prior application of cell-permeable hydroxyl radical scavengers, suggesting a role for this reactive oxygen species as an important intracellular signal mediating the first step. In these present experiments, we evaluated the potential role of reduction-oxidation mechanisms in the heat shock activation step. The thiol reducing agents reduced glutathione (GSH), n-acetylcysteine (NAC), and dithiothreitol (DTT) were evaluated for their ability to block programmed cell death in LPS-primed porcine aortic endothelial cells. Both DTT and NAC, agents that augment intracellular reduced glutathione levels, were protective against cell death when applied prior to heat shock induction with sodium arsenite (As) in endothelial cells treated previously with LPS. The less cell permeable agent GSH was not protective. Delayed application of DTT or NAC could block progression to cell death for up to 1.5 h after initiation of the heat shock response with As. These data show that heat shock-induced programmed cell death in LPS-primed endothelial cells can be arrested, at least in its early stages, by agents that augment or stabilize the reducing potential of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcysteine↗
PubMed1994

Origin of multiple bands of proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis--intermolecular disulphide cross-linking due to the presence of oxidizing components in the reducing agent.

The presence of oxidized products in the reducing agent used in sodium dodecyl sulphate-polyacrylamide gel electrophoresis is shown to yield multiple bands from otherwise homogeneous RNase A. The role of oxidized products in generating multiple bands is elucidated by using varying proportions of oxidized and reduced glutathione in a mixture as a reducing agent.

Cross-Linking Reagents↗
PubMed1997

Ascorbic acid and reducing agents regulate the fates and functions of S-nitrosothiols.

Although S-nitrosoglutathione (GS-NO) and other S-nitrosothiols (RS-NO) exhibit activity attributable to nitric oxide (NO), the dynamic aspects of their metabolism remain to be elucidated. To determine the fates and functions of RS-NO, the stability of GS-NO was analyzed in plasma, and various fractions of liver and kidney. GS-NO was fairly stable under physiological conditions in plasma and buffer solutions. However, GS-NO was rapidly decomposed in the presence of either homogenates of rat liver and kidney or their supernatant fractions. The ability of the supernatants to decompose GS-NO remained unchanged after the removal of proteins and large molecular weight compounds. Physiological levels of reducing agents, such as reduced glutathione (GSH), ascorbic acid (AsA), and cysteine, also enhanced the decomposition of RS-NO; the order of their potency was AsA > cysteine >GSH. Considering their intra-cellular concentrations and potency, AsA might principally be responsible for the enhanced decomposition of GS-NO. NO, GS-NO, and related RS-NO inhibited the respiration of Ehrlich ascites tumor cells. The inhibitory effect of GS-NO was enhanced by the reducing agents (cysteine>AsA>GSH). Intravenously administered GS-NO exhibited a depressor action through some ascorbic acid enhancable mechanism. Thus, the metabolism and biological function of GS-NO and related RS-NO might be affected by AsA and other reducing agents.

Animals↗
PubMed1992

Alteration of cell cycle kinetics by reducing agents in human peripheral blood lymphocytes from adult and senescent donors.

For improving cell proliferation reducing agents are routinely used as medium supplements in murine cell cultures, however, they are rarely used for human peripheral blood lymphocytes (PBLs). Data on changes in cell kinetics induced by reducing agents are not available. Here cell kinetic alterations induced by reducing agents in human lymphocytes are revealed by applying flow cytometric BrdUrd/Hoechst cell cycle analysis and by using the exit kinetic model of Smith and Martin. Applying alpha-thioglycerol (a-TG) as a model compound it was shown that the major cell kinetic effect is a shortening of the mean duration of the G0/G1 phase. The minimum G0/G1 phase duration and the percentage of the non-cycling G0/G1 cell fraction decrease only slightly. Moreover, a lower number of PBL's are arrested in the G2/M phase of the 1st cell cycle. The durations of the S and G2/M phase in the 1st and G1 phase in the 2nd cycle are not affected. These cell kinetic effects are identical for lymphocytes from both adult and senescent donors. The supplementation of the cell cultures with recombinant IL-2 did not induce similar cell kinetic alterations compared with a-TG. This indicates that the variation of the cell cycle progression factor IL-2 is not solely responsible for improvement of the cell activation process in the G0/G1 phase.

Aging↗
PubMed1977

Amorphous ferrous sulfide as a reducing agent for culture of anaerobes.

Amorphous ferrous sulfide, prepared by reacting ferrous ammonium sulfate and sodium sulfide, is an excellent reducing agent for the culture of anaerobes. It reduces resazurin and reacts much more rapidly with O2 than does either soluble sulfide (HS)- or cysteine. One of the end products of the oxidation of ferrous sulfide with O2 is red and serves as an indicator for the oxygen contamination of a culture medium. Amorphous ferrous sulfide served as a suitable reducing agent for the growth of species of Methanobacterium or Clostridium. Its use is recommended for enrichment or culture of anaerobes (e.g. autotrophs, fermentative organisms) from sediments and other habitats were organic reducing agents are undesirable and where soluble sulfide might be toxic.

Anaerobiosis↗
PubMed2006

Electrophoretic separation of human skeletal muscle myosin heavy chain isoforms: the importance of reducing agents.

An electrophoretic protocol previously used for the separation of rat myosin heavy chain (MHC) isoforms was slightly modified to improve the separation of human MHC isoforms in both large and minigel systems. The addition of reducing agents (beta-mercaptoethanol or dithiothreitol) to the top running buffer (TRB) radically improved separated MHC isoform resolution and the intensity of electrophoretic runs lasting longer than 5 h. In minigel systems, the MHC isoforms could be separated in as little as 5 h. The improved resolution of bands with the inclusion of reducing agents to the TRB facilitated the identification of clear boundaries for densitometric quantification of relative MHC isoform content, particularly for MHC IIa and MHC IIx. No significant effect of these reducing agents added to the TRB was observed for runs lasting only 100 min. Thus the inclusion of reducing agents in the TRB is essential for long electrophoretic runs, usually when separating large molecular mass proteins.

Electrophoresis↗
PubMed1982

Effects of oxygen tension and reducing agents on sensitivity of Giardia lamblia to metronidazole in vitro.

The effects of oxygen tension and reducing agents upon the sensitivity of Giardia lamblia to metronidazole in vitro were determined using two different assays. First, the parasites were exposed to drug under high or low oxygen tension in liquid medium with or without fresh cysteine and ascorbic acid. Survival was determined by colony assay. Second, the effect of thiol reducing agents was measured directly by colony assay in semi-solid media (low pO2 conditions). Trophozoites were strikingly (greater than 100-fold) less sensitive to metronidazole under the aerobic condition of the first assay. In contrast, the reducing agents had much lesser effects (2- to 5-fold) depending upon the assay. Finally, G. lamblia isolated from a patient treated with metronidazole unsuccessfully four times showed similar metronidazole sensitivity to a standard strain under both aerobic and reduced O2 tension conditions.

Animals↗
PubMed1995

Activation of NF-kappa B and elevation of MnSOD gene expression by thiol reducing agents in lung adenocarcinoma (A549) cells.

The effect of reducing agents, including N-acetylcysteine (NAC), dithiothreitol (DTT), and 2-mercaptoethanol (2-ME) on nuclear transcription factor-kappa B (NF-kappa B) activation and manganese superoxide dismutase (MnSOD) expression was investigated in a pulmonary adenocarcinoma (A549) cell line. NAC, DTT, and 2-ME each activated the transcription factor NF-kappa B and increased steady-state levels of MnSOD mRNA and enzyme activity in these cells. In addition, NAC, DTT, and 2-ME increased chloramphenicol acetyltransferase (CAT) activity in cells transfected with a construct containing the CAT gene under the control of the rat MnSOD promoter. SOD and catalase (500 U/ml) plus ethanol (1 mM) did not inhibit activation of NF-kappa B or elevation of steady-state MnSOD mRNA levels by NAC, DTT, or 2-ME. Controls in which comparable amounts of O2-. to those produced by thiols were generated by hypoxanthine and xanthine oxidase, or in which H2O2 was added directly, had neither activated NF-kappa B nor elevated MnSOD mRNA. This shows that reactive oxygen intermediates, which may be formed during autooxidation, may not contribute to activation of NF-kappa B. Because the MnSOD promoter also contains potential binding sites for other transcription factors, such as promoter-selective transcription factor-1 (SP-1), activator protein-1 (AP-1), AP-2, adenosine 3',5'-cyclic monophosphate-regulator element binding factor (CREB), and transcription factor IID complex (TFIID), the effect of thiols on their activation also were evaluated. In contrast to findings with NF-kappa B, there was only minor activation of AP-1 by thiols, and none of the other transcription factors were activated by thiols. AP-1 activation was inhibited by catalase (500 U/ml) plus SOD plus ethanol (1 mM). Addition of 700 microM H2O2 also activated AP-1, and catalase at 500 U/ml prevented this activation. This indicates that H2O2 produced as a result of autooxidation of thiols can activate AP-1 but not NF-kappa B. Thus a close association between exposure to reducing agents, activation of NF-kappa B, and elevation of MnSOD gene expression is demonstrated.

Acetylcysteine↗
PubMed1986

Sulfhydryl reducing agents and shape regulation in human erythrocytes.

Metabolic crenation of red cells is reversible; on addition of nutrients, echinocytes recover the normal discoid shape. When the shape recovery takes place in the presence of reducing agents such as dithiothreitol (DTT), morphological change continues until the cells are stomatocytic. The degree of stomatocytosis varies, depending on the cell morphology when the nutrients and reducing agent are added. DTT has minimal effect on the shape of normal discocytes, but in its presence, mildly echinocytic cells become slightly cupped and advanced-stage echinocytes become severely stomatocytic. DTT must be present continuously for development and retention of stomatocytosis; echinocytes preincubated with or metabolically depleted in DTT do not become stomatocytic when supplemented in the absence of DTT, and DTT-induced stomatocytes revert to discocytes when the reducing agent is removed. DTT has no effect on adenosine triphosphate synthesis or equilibrium cell glutathione levels, and the induced stomatocytosis is not inhibited by excluding oxygen from cells during depletion. Spectrin phosphorylation and phosphate turnover are not affected by DTT. The echinocyte-to-discocyte transformation coincides with phosphorylation of membrane inner monolayer lipids (diacylglycerol to phosphatidic acid and phosphatidylinositol to phosphatidylinositol-4,5-bisphosphate). Overphosphorylation of these phospholipids is not responsible for the exaggerated shape recovery seen with reducing agents; phosphorylation of inner monolayer lipids proceeds identically in the presence and absence of DTT.

Adenosine Triphosphate↗
PubMed2004

Photoinduced interaction of riboflavin dye with different reducing agents in aqueous and liposome media.

The photoelectrochemical and spectral studies of riboflavin have been carried out in aqueous and phosphatidylcholine (PC) liposome media in presence of different reducing agents such as I-, Br-, Cl-, Fe2+, Fe(CN)6(4-) and Cu+. The results from both the studies support the photoinduced electron transfer from the reducing agent to the excited riboflavin dye. Moreover, a good correlation between photovoltages/Stern-Volmer quenching constants versus reduction potentials of the reducing agents also confirms the above electron transfer in the photoexcited state. An alternative method has been developed to determine the Stern-Volmer quenching constant.

Coloring Agents↗
PubMed1980

Effect of reducing agents on the solubilization of renal sodium and potassium dependent ATPase with detergent.

A high concentration (0.5 M) of a reducing agent such as dithiothreitol or 2-mercaptoethanol inactivated both crude (microsomal) and purified preparations of Na+,K+-ATPase only in the presence of detergent. The concentration of SDS needed for inactivation by a reducing agent was low (around 0.3 mg/ml), and was similar to that required for activation in the absence of reduction. The subunits of the enzyme after inactivation with a reducing agent were not sedimented by centrifugation at 100,000 x g for 1 h. Electron microscopic observation of the inactivated enzyme revealed that membranous structures were dominant in the pellet obtained by centrifugation, whereas many particles were present in the supernatant. These results suggest that Na+,K+-ATPase was inactivated by reduction of disulfide bond(s) embedded within the lipid bilayer in the presence of detergent, and that the subunits of the resulting enzyme were no longer bound to the membrane.

Animals↗
PubMed2001

Photophysics of thionine dye in aqueous and liposome media in presence of different reducing agents.

The photoelectrochemical and spectral (both absorption and fluorescence) studies of thionine, a cationic phenothiazine dye, have been carried out in aqueous and phosphatidylcholine liposome media in the presence of different reducing agents, such as I(-), Br(-), Cl(-) and Fe(2+). The results show that the photovoltage generation from photoelectrochemical studies and Stern-Volmer quenching constant studied by fluorescence quenching support the photoinduced electron transfer from the reducing agent to the singlet excited thionine dye. Moreover, a good correlation between photovoltages/Stern-Volmer quenching constants vs. reduction potentials of the reducing agents also confirms the above electron transfer in the photoexcited state.

Liposomes↗
PubMed2000

Comparison of different reducing agents for enhanced detection of heat-injured Listeria monocytogenes.

The effect of different reducing agents (L-cysteine, Oxyrase, and Enterococcus faecium) and their combinations on the detection of heat-injured (62.8 degrees C, 7.5 min or 10 min) Listeria monocytogenes was examined. The incorporation of L-Cysteine (0.5 g/liter) yielded higher percentage detection than any of the other reducing agents (P < 0.05). The combination of Oxyrase (10 U/ml) and E. faecium (10(3) CFU/ml) synergistically enhanced the detection of L. monocytogenes heat-injured for 10 min at 62.8 degrees C (P < 0.05). Simultaneous addition of L-cysteine (0.5 g/liter) and Oxyrase (10 U/ml) significantly lowered the recovery of heat-injured L. monocytogenes (P < 0.05). Higher activities of Oxyrase (50 U/ml) inhibited the detection of heat-injured L. monocytogenes (P < 0.05). The rates of depletion of relative percentage O2 were in the order: L-cysteine (0.5 g/liter; 6.63%/ min) > Oxyrase (10 U/ml; 5.00%/min) > E. faecium (10(8) CFU/ml; 1.66%/min) > E. faecium (10(3) CFU/ml; 0.20%/min). The final levels of redox potential (Eh) achieved were -110.5 mV, -100 mV, -83.5 mV, and -25 mV for E. faecium (10(8) CFU/ml), L-cysteine, Oxyrase, and E. faecium (10(3) CFU/ml), respectively.

Animals↗
PubMed2006

Effects of oxidizing and reducing agents on ovine pulmonary artery responses to nitric oxide donors, sodium nitroprusside and 3-morpholino-sydnonimine.

Nitrovasodilators-sodium nitroprusside (SNP; 10(-9)-10(-4) M) and 3-morpholino-sydnonimine (SIN-1; 10(-9)-10(-4) M) produced concentration-dependent relaxation of the fourth generation sheep pulmonary artery, preconstricted with 5-hydroxytryptamine (1 microM). Oxidizing agents [oxidized glutathione (GSSG, 1 mM) and CuSO4 (5 and 20 microM)] and reducing agents [dithiothreitol (DTT, 0.1 mM), ascorbic acid (1 mM) and reduced glutathione (GSH, 1 mM)] caused opposite effects on nitric oxide (NO)-induced vasodilation in the artery. Ascorbic acid and GSH potentiated the NO responses, while GSSG and CuSO4 inhibited relaxation caused by the nitrovasodilators. DTT, however, reduced the relaxant potency and efficacy of SNP and SIN-1. Pretreatment of the pulmonary artery strips with DTT (0.1 mM) inhibited SNP (10 microM)-induced Na(+)-K(+)-ATPase activity, while ascorbic acid (1 mM) and GSH (1 mM) had no effect either on basal or SNP (10 microM)-stimulated 86Rb uptake, an index of Na(+)-K(+)-ATPase activity, in ovine pulmonary artery. The results suggest that reducing agents like ascorbic acid may have beneficial effect in improving the vascular function under oxidative stress.

Animals↗
PubMed1985

Identification of gallbladder mucin-bilirubin complex in human cholesterol gallstone matrix. Effects of reducing agents on in vitro dissolution of matrix and intact gallstones.

The goals of this study were to isolate and characterize the nonlipid matrix of human cholesterol gallstones. The lipid portion of gallstones was dissolved in ethanol/ether, leaving an insoluble, granular, brown-black matrix that constituted 12.5% of solitary large stones and 3.5% of multiple small stones. The matrix was partially solubilized by sonication and studied by exclusion gel chromatography and density gradient ultracentrifugation. On Sepharose 2B column chromatography, bile pigment eluted with glycoprotein in the void volume, suggesting the presence of a high molecular weight complex (Mr greater than 2 X 10(6)). The identity of mucin in this complex was confirmed by its typical buoyant density during ultracentrifugation. The major bile pigments in the matrix were identified as bilirubin (84%) and bilirubin monoglucuronide (15%) by thin-layer chromatography. Because of their ability to solubilize mucin-type glycoproteins, we tested the ability of the reducing agents 2-mercaptoethanol (2ME) and N-acetylcysteine (NAcCys) to solubilize gallstone matrix. Both reducing agents caused a two- to threefold enhancement of matrix dissolution after 4 d compared to aqueous buffer alone (P less than 0.01). Sepharose 2B chromatography revealed that 2ME released a high molecular weight mucin-bilirubin complex as well as unbound pigment from the insoluble matrix. We also tested the effect of reducing agents on dissolution of matched cholesterol gallstones by monooctanoin, a cholesterol solvent. Both 2ME and NAcCys significantly accelerated gallstone dissolution in monooctanoin. Matched human cholesterol stones (n = 10) incubated for 4 d in monooctanoin plus either 2ME or NAcCys (1 M final concentration) weighed approximately half as much (P less than 0.01 for each) as stones incubated in monooctanoin alone. This study describes, for the first time, the isolation of a bilirubin-mucin complex in the insoluble matrix of human cholesterol gallstones. The ability of reducing agents to dissolve the matrix and thereby accelerate gallstone dissolution by monooctanoin in vitro may be relevant to gallstone dissolution in humans.

Acetylcysteine↗
PubMed1997

Evidence for a specific interaction of vitronectin with arginine: effects of reducing agents on the expression of functional domains and immunoepitopes.

Vitronectin (Vn) circulates in plasma primarily in the native, monomeric form, whereas platelet-associated Vn in conformationally altered and multimeric. Here, we report that denatured Vn specifically binds to L-Arg, whereas the L-Arg binding site is cryptic in the native form of Vn. In addition, combined treatment of disulfide-linked Vn multimers with L-Arg, urea, and reducing agent results in the formation of disperse oligomers with reduced expression of denaturation-sensitive epitopes. These results suggest that L-Arg modulates the partitioning between monomeric and multimeric Vn species and that L-Arg affinity chromatography can be employed to test for exposure of conformationally sensitive binding sites in Vn. The effects of denaturation on the exposure of conformationally sensitive epitopes in the N-terminus of Vn is controversial. Treatment of Vn with reducing agents abolished type 1 plasminogen activator inhibitor and antibody binding to the highly disulfide-linked N-terminal somatomedin B domain (amino acids 1 to 51), whereas epitopes located in the connecting region/first hemopexin-like repeat (amino acids 52 to 239) and the glycosaminoglycan binding domain (amino acids 343-379) were not affected. These observations indicate that appropriate disulfide-linkage of the N-terminal somatomedin B domain is required for ligand binding and that published differences on the effects of denaturation on the expression of binding sites are probably due to the use of reducing agents in the denaturation process.

Antibodies, Monoclonal↗
PubMed1997

Identification in the mu-opioid receptor of cysteine residues responsible for inactivation of ligand binding by thiol alkylating and reducing agents.

Inactivation by thiol reducing and alkylating agents of ligand binding to the human mu-opioid receptor was examined. Dithiothreitol reduced the number of [3H]diprenorphine binding sites. Replacement by seryl residues of either C142 or C219 in extracellular loops 1 and 2 of the mu receptor resulted in a complete loss of opioid binding. A disulfide bound linking C142 to C219 may thus be essential to maintain a functional conformation of the receptor. We also demonstrated that inactivation of ligand binding upon alkylation by N-ethylmaleimide occurred at two sites. Alteration of the more sensitive (IC50 = 20 microM) did not modify antagonists binding but decreased agonist affinity almost 10-fold. Modification of the less reactive site (IC50 = 2 mM) decreased the number of both agonist and antagonist binding sites. The alkylation site of higher sensitivity to N-ethylmaleimide was shown by mutagenesis experiments to be constituted of both C81 and C332 in transmembrane domains 1 and 7 of the mu-opioid receptor.

Alkylating Agents↗