• BMB Reports
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• v.55 no.3
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• pp.154-159
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• 2022

Protein S-glutathionylation is a reversible post-translational modification on cysteine residues forming a mixed disulfide with glutathione. S-glutathionylation, not only protects proteins from oxidation but also regulates the functions of proteins involved in various cellular signaling pathways. In this study, we developed a method for the detection of S-glutathionylated proteins (ProSSG) using eosin-glutathione (E-GSH) and mouse glutaredoxin 1 (mGrx1). ProSSG was efficiently and specifically labeled with E-GSH to form ProSSG-E via thiol-disulfide exchange. ProSSG-E was readily luminescent allowing the detection of ProSSG with semi-quantitative determination. In addition, a deglutathionylation enzyme mGrx1 specifically released E-GSH from ProSSG-E, which increased fluorescence allowing a sensitive determination of ProSSG levels. Application of the method to the adipocyte differentiation of 3T3-L1 cells showed specific detection of ProSSG and its increase upon differentiation induction, which was consistent with the result obtained by conventional immunoblot analysis, but with greater specificity and sensitivity.

• Microbiology and Biotechnology Letters
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• v.26 no.6
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• pp.483-489
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• 1998

We carried out the expression and characterization of yeast thioredoxin system including thioredexin 1 (Trx1), Trx2, thioredoxin reductase (TR), and a novel thioredoxin (Trx3), which was reported in the data base of Saccharomyces genome. The Trx1, 2 and TR were expressed as soluble proteins in E. coli and the sizes of purified proteins were equal to the reported their molecular weights. The expressed Trx3 was found in both soluble fraction and precipitate. The size of Trx3 purified from soluble fraction of E. coli crude extracts was estimated as 14 kDa on SDS-PAGE instead of 18 kDa for Trx3 in precipitate. N-terminal amino acid sequence of the small size of purified Trx3 from soluble fraction was analyzed as FQSSYTS which is correspond to the sequence from 20 to 26 for Trx3. Trx3 together with thioredoxin reductase and NADPH was able to reduce the disulfide bridge of insulin and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Trx3 stimulated the antioxidant effect of thioredoxin peroxidase 1 (TPx1) which inhibited inactivation of glutamine synthetase (GS) in dithiothreitol (DTT) containing metal catalyzed oxidation system. The stimulation effect of Trx3 was 10% of the effect of either Trx1 or Trx2. In addition, Trx3 could reduce the disulfide of TPx to thiol, so that the TPx had thioredoxin dependant peroxidase activity. In western blotting analysis, antibodies against purified Trx3 did not cross-react with crude extracts of yeast, purified Trx1, and Trx2 proteins. But, in PCR reaction using the cDNA library of yeast as a template, gene encoding of trx3 was amplified.

• Biomedical Science Letters
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• v.4 no.1
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• pp.1-9
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• 1998

A Corynebacterium diphtheriae iron-repressible gene dirA, that was homologous to TSA of Saccharomyces cerevisiae and AhpC subunit of Salmonella typhimurium alkyl hydroperoxide reductase, was amplified with PCR and expressed in E. coli. The DirA purified from the transformed E. coli crude extracts prevented the inactivation of enzyme caused by metal-catalyzed oxidation (MCO) system containing thiols but not by ascorbate/Fe$^{3+}$/$O_2$ MCO system. The DirA concentration, which inhibited the inactivation of glutamine synthetase by 50% (IC$_{50}$) against MCO system, was 0.12 mg/ml. The multimeric forms of DirA were converted to the monomeric form in SDS-PAGE under the thioredoxin system comprised of NADPH, Saccharomyces cerevisiae thioredoxin reductase, and thioredoxin. Also, DirA showed thioredoxin dependent peroxidase activity. All of these results were consistent with the characteristics of a thiol specific antioxidant (TSA) protein having two conserved cysteine residues.

• Journal of Animal Science and Technology
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• v.58 no.6
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• pp.23.1-23.17
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• 2016

Background: The functionality of myofibrillar proteins is a major factor influencing the quality attributes of muscle foods. Nonetheless, the relationships between muscle type and oxidative changes in chevon during ageing are meagrely elucidated. Postmortem changes in antioxidant status and physicochemical properties of glycolytic gluteus medius (GM) and oxidative infraspinatus (IS) muscles in goats were compared. Methods: Twenty Boer bucks (9-10 months old, body weight of $36.9{\pm}0.725kg$) were slaughtered and the carcasses were subjected to chill storage ($4{\pm}0.5^{\circ}C$). Analyses were conducted on GM and IS muscles sampled on 0, 1, 4 and 7 d postmortem. Results: Chill storage did not affect the antioxidant enzyme activities in both muscles. The IS had greater (P < 0.05) superoxide dismutase and catalase activities than GM. Carotenoid and tocopherol contents did not differ between muscles but decreased (P < 0.05) over storage. The IS had higher (P < 0.05) glycogen and ultimate pH and lower (P < 0.05) shear force and cooking loss than GM. The carbonyl content, % metmyoglobin, drip loss and TBARS increased (P <0.05) while free thiol, metmyoglobin reducing activity (MRA), shear force and myoglobin decreased (P < 0.05) over storage. Muscle type had no effect (P > 0.05) on free thiol, MRA and TBARS. The GM had lower (P < 0.05) redness on d 0 and 1 than IS while the IS had greater carbonyl, % metmyoglobin and drip loss than GM on d 7. The reflective density of slow myosin heavy chain (MHC) was higher (P < 0.05) while the density of fast MHC and actin was lower (P < 0.05) in IS than GM. Regardless of muscle type, the density of MHC decreased (P < 0.05) while that of actin was stable over storage. Nonetheless, the degradation of fast and slow MHC was greater (P < 0.05) in IS than GM. Muscle type had no effect (P > 0.05) on consumer preference for flavour, juiciness and overall acceptability. However, IS had higher (P < 0.05) tenderness score than GM on d 1 and 4 postmortem. Intramuscular fat was higher (P< 0.05) in IS compared with GM. Fatty acid composition did not differ between the muscles. However, GM had lower (P < 0.05) n-6/n-3 ratio than IS. The n-3 and n-6 PUFA declined (P < 0.05) while the SFA increased (P < 0.05) over storage. Conclusion: The changes in myofibrillar proteins and physicochemical properties of goat meat during postmortem chill storage are muscle-dependent.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.74-78
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• 2003

Multi-channel images of 11-MUA and 11-MUOH self-assembled monolayers were obtained by using two-dimensional surface plasmon resonance (SPR) absorption. Patterning process was simplified by exploiting direct photo-oxidation of thiol bonding (photolysis) instead of conventional photolithography. Sharper images were resolved by using a white light source in combination with a narrow bandpass filter in the visible region, minimizing the diffraction patterns on the images. The line profile calibration of the image contrast caused by different resonance conditions at each points on the sensor surface (at a fixed incident angle) enables us to discriminate the monolayer thickness in sub-nanometer scale. Furthermore, there is no signal degradation such as photo bleaching or quenching which are common in the detection methods based on the fluorescence.

• International Journal of Oral Biology
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• v.39 no.4
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• pp.229-236
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• 2014

Reactive oxygen species (ROS) and nitrogen species (RNS) are implicated in cellular signaling processes and as a cause of oxidative stress. Recent studies indicate that ROS and RNS are important signaling molecules involved in nociceptive transmission. Xanthine oxidase (XO) system is a well-known system for superoxide anions ($O{_2}^{{\cdot}_-}$) generation, and sodium nitroprusside (SNP) is a representative nitric oxide (NO) donor. Patch clamp recording in spinal slices was used to investigate the role of $O{_2}^{{\cdot}_-}$ and NO on substantia gelatinosa (SG) neuronal excitability. Application of xanthine and xanthine oxidase (X/XO) compound induced membrane depolarization. Low concentration SNP ($10{\mu}M$) induced depolarization of the membrane, whereas high concentration SNP (1 mM) evoked membrane hyperpolarization. These responses were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger). Addition of thapsigargin to an external calcium free solution for blocking synaptic transmission, led to significantly decreased X/XO-induced responses. Additionally, X/XO and SNP-induced responses were unchanged in the presence of intracellular applied PBN, indicative of the involvement of presynaptic action. Inclusion of GDP-${\beta}$-S or suramin (G protein inhibitors) in the patch pipette decreased SNP-induced responses, whereas it failed to decrease X/XO-induced responses. Pretreatment with n-ethylmaleimide (NEM; thiol-alkylating agent) decreased the effects of SNP, suggesting that these responses were mediated by direct oxidation of channel protein, whereas X/XO-induced responses were unchanged. These data suggested that ROS and RNS play distinct roles in the regulation of the membrane excitability of SG neurons related to the pain transmission.

• Journal of Microbiology and Biotechnology
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• v.28 no.7
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• pp.1209-1216
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• 2018

This study aimed to evaluate the antimicrobial activity of 2-phenylethynyl-butyltellurium (PEBT) in Escherichia coli and the relation to its pro-oxidant effect. For this, we carried out the disk diffusion test, minimum inhibitory concentration (MIC) assay, and survival curve analysis. We also measured the level of extracellular reactive oxygen species (ROS), activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), and level of non-protein thiols (NPSH). PEBT at 1.28 and 0.128 mg/disk exhibited antimicrobial capability in the disk diffusion test, with an MIC value of 1.92 mg/ml, whereas PEBT at 0.96, 1.92, and 3.84 mg/ml inhibited bacterial growth after a 9-h exposure. PEBT at 3.84, 1.92, and 0.96 mg/ml increased extracellular ROS production, decreased the intracellular NPSH level, and reduced the SOD and CAT activities. Glutathione or ascorbic acid in the medium protected the bacterial cells from the antimicrobial effect of PEBT. In conclusion, PEBT exhibited antimicrobial activity against E. coli, involving the generation of ROS, oxidation of NPSH, and reduction of the antioxidant defenses in the bacterial cells.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.1
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• pp.25-36
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• 2022

To identify the effect and mechanism of carbon monoxide (CO) on delayed rectifier K⁺ currents (I_K) of human cardiac fibroblasts (HCFs), we used the wholecell mode patch-clamp technique. Application of CO delivered by carbon monoxidereleasing molecule-3 (CORM3) increased the amplitude of outward K⁺ currents, and diphenyl phosphine oxide-1 (a specific I_K blocker) inhibited the currents. CORM3-induced augmentation was blocked by pretreatment with nitric oxide synthase blockers (L-NG-monomethyl arginine citrate and L-NG-nitro arginine methyl ester). Pretreatment with KT5823 (a protein kinas G blocker), 1H-[1,-2,-4] oxadiazolo-[4,-3-a] quinoxalin-1-on (ODQ, a soluble guanylate cyclase blocker), KT5720 (a protein kinase A blocker), and SQ22536 (an adenylate cyclase blocker) blocked the CORM3 stimulating effect on I_K. In addition, pretreatment with SB239063 (a p38 mitogen-activated protein kinase [MAPK] blocker) and PD98059 (a p44/42 MAPK blocker) also blocked the CORM3's effect on the currents. When testing the involvement of S-nitrosylation, pretreatment of N-ethylmaleimide (a thiol-alkylating reagent) blocked CO-induced I_K activation and DL-dithiothreitol (a reducing agent) reversed this effect. Pretreatment with 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)-21H,23H porphyrin manganese (III) pentachloride and manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (superoxide dismutase mimetics), diphenyleneiodonium chloride (an NADPH oxidase blocker), or allopurinol (a xanthine oxidase blocker) also inhibited CO-induced I_K activation. These results suggest that CO enhances I_K in HCFs through the nitric oxide, phosphorylation by protein kinase G, protein kinase A, and MAPK, S-nitrosylation and reduction/oxidation (redox) signaling pathways.

• Applied Chemistry for Engineering
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• v.24 no.5
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• pp.551-557
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• 2013

In the present study, pristine carbon nanotube (p-CNT) and thiolated carbon naotube (t-CNT) electrodes were investigated to improve their detectabilities for cadmium (Cd) and lead (Pb). In addition, we evaluate which reaction mechanism is used when the electrolyte contains both Cd and Pb metals. Square wave stripping was employed for analyzing the sensitivity for the metals. A frequency of 30 Hz, a deposition potential of -1.2 V vs. Ag/AgCl and a deposition time of 300 s were used as optimal SWSV parameters. t-CNT electrodes show the better sensitivity for both Cd and Pb metals than that of p-CNT electrodes. In case of Cd, sensitivities of p-CNT and t-CNT electrodes were $3.1{\mu}A/{\mu}M$ and $4.6{\mu}A/{\mu}M$, respectively, while the sensitivities for Pb were $6.5{\mu}A/{\mu}M$ (p-CNT) and $9.9{\mu}A/{\mu}M$ (t-CNT), respectively. The better sensitivity of p-CNT electrodes is due to the enhancement in the reaction rate of metal ions that are facilitated by thiol groups attached on the surface of CNT. When sensitivity was measured for the detection of Cd and Pb metals present simultaneously in the electrolyte, Pb indicates better sensitivity than Cd irrespective of electrode types. It is ascribed to the low standard electrode potential of Pb, which then promotes the possibility of oxidation reaction of the Pb metal ions. In turn, the Pb metal ions are deposited on the electrode surface faster than that of Cd metal ions and cover the electrode surface during deposition step, and thus Pb metals that cover the large portion of the surface are more easily stripped than that of Cd metals during stripping step.

• Journal of Plant Biotechnology
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• v.33 no.1
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• pp.1-9
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• 2006

Peroxiredoxins (Prxs) are ubiquitously distributed and play important functions in diverse cellular signaling systems. The proteins are largely classified into three groups, such as typical 2-Cys Prx, atypical 2-Cys Prx, and 1-Cys Prx, that are distinguished by their catalytic mechanisms and number of Cys residues. From the three classes of Prxs, the typical 2-Cys Prx containing the two-conserved Cys residues at its N-terminus and C-terminus catalyzes $H_2O_2$ with the use of thioredoxin (Trx) as an electron donor. During the catalytic cycle, the N-terminal Cys residue undergoes a peroxide-dependent oxidation to sulfenic acid, which can be further oxidized to sulfinic acid at the presence of high concentrations of $H_2O_2$ and a Trx system containing Trx, Trx reductase, and NADPH. The sulfinic acid form of 2-Cys Prx is reduced by the action of sulfiredoxin which requires ATP as an energy source. Under the strong oxidative or heat shock stress conditions, 2-Cys Prx in eukaryotes rapidly switches its protein structure from low-molecular-weight species to high-molecular-weight protein structures. In accordance with its structural changes, the protein concomitantly triggers functional switching from a peroxidase to a molecular chaperone, which can protect its substrate denaturation from external stress. In addition to its N-terminal active site, the C-terminal domain including 'YF-motif' of 2-Cys Prx plays a critical role in the structural changes. Therefore, the C-terminal truncated 2-Cys Prxs are not able to regulate their protein structures and highly resistant to $H_2O_2$-dependent hyperoxidation, suggesting that the reaction is guided by the peroxidatic Cys residue. Based on the results, it may be concluded that the peroxidatic Cys of 2-Cys Prx acts as an '$H_2O_2$-sensor' in the cells. The oxidative stress-dependent regulation of 2-Cys Prx provides a means of defense systems in cells to adapt stress conditions by activating intracellular defense signaling pathways. Particularly, 2-Cys Prxs in plants are localized in chloroplasts with a dynamic protein structure. The protein undergoes conformational changes again oxidative stress. Depending on a redox-potential of the chloroplasts, the plant 2-Cys Prx forms super-molecular weight protein structures, which attach to the thylakoid membranes in a reversible manner.