Ammonium (NH4 +) serves as a nitrogen source, but its elevated levels can hinder plant growth and production. Excess NH4 + with α-ketoglutarate is assimilated into glutamate, a precursor of proline and glutathione (GSH). This study aimed to investigate the effects of excessive NH4 + on the regulation of proline and GSH synthesis. Detached leaves from oilseed rape (Brassica napus L.) were fed with 0, 50, 100, 500, and 1000 mM NH4Cl for 16 h. As the NH4 + concentrations increased, the leaves exhibited progressive wilting and yellowing. Furthermore, total carotenoid and chlorophyll concentrations declined in response to all NH4 + treatments, with the lowest levels observed in 1000 mM NH4 + treatment. Hydrogen peroxide (H2O2) concentration showed a minor increase at low NH4 + concentration (50 and 100 mM) treatments but a significant increase at high NH4 + (500 and 1000 mM), which was consistent with the localization of H2O2. Amino acid concentrations increased with increasing in NH4 + concentration, while the protein concentration displayed the opposite trend. Proline and cysteine concentrations exhibited a gradual increase in response to increasing NH4 + concentrations. However, GSH concentrations rose only in the 50 mM NH4 + treatment and decreased in the 500 and 1000 mM NH4 + treatments. These results indicate that excessive NH4 + is primarily assimilated into proline, while GSH synthesis is adversely affected.

본 연구는 식물검역 분야에서 주요하게 사용되고 있는 메틸브로마이드 훈증제로 인해 발생하는 약해를 저감하기 위한 물질을 모델식물인 애기장대를 이용하여 스크리닝하였다. 사전연구를 통하여 메틸브로마이드 훈증제의 식물 독성 메커니즘으로 활성산소발생와 식물 성장 호르몬인 옥신의 식물체 내 분배억제효과가 발생하는 것을 바탕으로 하여, 약해 저감물질후보군으로 활성산소를 제거하는 역할을 하는 ROS scavenger 2종 (NAC, GSH)과 옥신을 훈증제 처리 전 애기장대에 처리한 후 약해의 저감 정도를 육안평가와 더불어 관련 유전자의 발현을 확인하였다. 연구 결과 메틸브로마이드에 의해 유도된 약해는 옥신보다는 활성산소를 저감시키는 물질후보군들에서 약해 저감효과가 나타났다. 이 중 GSH을 이용하여 농도구배하여 전처리하였을 때, 5 mM GSH 전처리 후 메틸브로마이드 훈증 시 약해 저감효과가 두드러졌다. GSH 전처리 시 식물체 내에 MBF1c와 HSP70 유전자 발현이 증가하는 것을 확인하였으며, 이는 메틸브로마이드 훈증으로 유도되는 약해를 방어하는 역할을 담당하였을 것이라고 평가된다. 따라서, 식물검역 훈증제 메틸브로마이드에 의해 발생하는 약해를 저감하는 데 GSH의 사용가능성을 평가하였으며, 이를 기반으로 다양한 식물체에 적용하여 수출입 시 약해로 인한 경제적 손실을 감소시킬 수 있기를 기대한다.

Zinc selenide (ZnSe) nanoparticles were synthesized in aqueous solution using glutathione (GSH) as a ligand. The influence of the ligand content, reaction temperature, and hydroxyl ion concentration (pH) on the fabrication of the ZnSe particles was investigated. The optical properties of the synthesized ZnSe particles were characterized using various analytical techniques. The nanoparticles absorbed UV-vis light in the range of 350-400 nm, which is shorter than the absorption wavelength of bulk ZnSe particles (460 nm). The lowest ligand concentration for achieving good light absorption and emission properties was 0.6 mmol. The reaction temperature had an impact on the emission properties; photoluminescence spectroscopic analysis showed that the photo-discharge characteristics were greatly enhanced at high temperatures. These discharge characteristics were also affected by the hydroxyl ion concentration in solution; at pH 13, sound emission characteristics were observed, even at a low temperature of 25oC. The manufactured nanoparticles showed excellent light absorption and emission properties, suggesting the possibility of fabricating ZnSe QDs in aqueous solutions at low temperatures.

The aim of present study was to investigate regulatory mechanism of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation of porcine oocytes. Basically, immature cumulus-oocyte complexes (COCs) were incubated for 22 h in IVM-I to which hormone was added, and then further incubated for 22 h in IVM-II without hormone. As a result, relative cumulus expansion was increased at 22 h after IVM and it was enhanced by treatment of ALA compared with control group (p < 0.05). During IVM process within 22 h, cAMP level in oocytes was decreased at 6 h (p < 0.05) and it was recovered at 12 h in ALA-treated group, while oocytes in control group recovered cAMP level at 22 h. In cumulus cells, it was reduced in all time point (p < 0.05) and ALA did not affect. Treatment of ALA enhanced metaphase-I (MI) and MII population of oocytes compared with oocytes in control group at 22 and 44 h, respectively (p < 0.05). Intracellular GSH levels in ALA group was increased at 22 and 44 h after IVM (p < 0.05), whereas it was increased in control group at 44 h after IVM (p < 0.05). In particular, the GSH in ALA-treated oocytes during 22 h of IVM was higher than control group at 22 h (p < 0.05). Lipid amount in oocytes from ALA group was higher than control group (p < 0.05). Treatment of ALA did not influence to absorption of glucose from medium. Cleavage and blastocyst formation of ALA-treated oocytes were enhanced compared with control group (p < 0.05). These findings suggest that supplementation of ALA could improve oocyte maturation and development competence through increasing GSH synthesis, lipid storage, and regulation of cAMP accumulation during early 22 h of IVM, and these might be mediated by cumulus expansion.

Glutathione S-transferase (GST) is a key gene involved in multiple stress tolerance in all living organisms, though it is still to be disclosed the gene function in teff grass [Eragrostis tef (Zucc.)Trotter].The objectives of this study were to clone and molecular characterization of GST gene in teff grass. We characterized GST1 from teff grass (EtGST1), it composed of a 645-bp open reading frame (ORF) that encoded 195 amino acid residue. Further, we transformed EtGST1 in E.coli BL21 (DE3) cells. This recombinant EtGST1 in E.coli BL21(DE3) induced at 37°C temperature. In addition, Growth of cells overexpressing EtGST1 rapidly increased in the presence of polyethylene glycol (5%), heat (46°C), NaCl (0.6%), and arsenic (1 mM) than that of cells harboring an empty vector. These results suggest that EtGST1 would be suitable candidate for improving tolerance in forages and/or grasses species against multiple abiotic stresses.

This study evaluated the effect of reduced glutathione (GSH) for the reduction of stress and inflammatory response in calves inoculated with foot-and-mouth disease (FMD) vaccine. Twenty-five calves were divided into five groups of 5 calves. The negative control (NC) did not receive any vaccination or drug treatment. The positive control (PC), GSH-25, GSH-50 and GSH-100 were intramuscularly injected with GSH at concentrations of 0, 25, 50 and 100 mg / 10 kg body weight (BW), respectively, for 3 days after FMD vaccination. On day 3, 5 and 7 post-treatment, the serum cortisol and tumor necrosis factor- α (TNF-α) levels in GSH-50 and GSH-100 were significantly decreased compared with those in PC (p < 0.05). However, there was no significant difference in the serum cortisol and TNF-α levels between GSH-100 and NC 3 and 5 days post-treatment, and between GSH-50, GSH-100, and NC 7 days post-treatment. The results from this study suggest that treatment of 50 mg / 10 kg BW GSH for 3 days is useful for the reduction of stress and inflammatory response caused by FMD vaccination in calves.

The present study was aimed to determine the effects of green tea extract (GTE) and beta-mercaptoethanol (β-ME) supplementation in boar sperm freezing extender on in vitro fertilization (IVF) and reactive oxygen species (ROS) and glutathione (GSH) levels of presumptive zygotes (PZs). Experimental groups were allocated into lactose egg yolk (LEY) without antioxidant (control), GTE (1,000 mg/l in LEY) and β-ME (50 μM in LEY). In freezing, spermatozoa extended with LEY were cooled to 5°C for 3 h and then kept at 5°C for 30 min following dilution with LEY containing 9% glycerol and 1.5% Equex STM. The final sperm concentration was 1 × 108/ml. Spermatozoa were loaded into straws and frozen in nitrogen vapor for 20 min. For IVF, oocytes were matured in NCSU-23 medium and co-cultured with spermatozoa following thawing at 37°C for 25 sec. At 12 h following IVF, IVF parameters (sperm penetration and monospermy) were evaluated. In addition, GSH and ROS levels of PZs were determined by Cell Tracker Blue CMF2HC and DCHFDA, respectively. IVF parameters did not show any significant difference among the experimental groups. GSH and ROS levels of PZs were not significantly different between groups. In conclusion, antioxidant supplementation in boar sperm freezing could not influence IVF parameters, ROS and GSH levels of PZs.

To increase the productivity of in vitro development, the antioxidants have been used for culture system of bovine oocytes and embryos. However, comparative studies on these molecules are rare and direct beneficial effects on blastocyst production cannot be discriminated for best results. The study was conducted to determine the influence of N-acetyl-L-cysteine (NAC), N-acetyl-L-cysteine amide (NACA), glutathione (GSH) and cysteamime (CYS) on maturation competence of COCs from GV to MII stage and productivity of blastocyst formation during in vitro fertilization and culture. There was no difference among maturation rates of oocytes to metaphase II with polar body with antioxidants for any of the treatment groups (p>0.05). However, the significant improvement on the rate of blastocysts (32.3±5.0%) was found in 0.1 mM CYS treatment than 0.3 mM NAC, 0.2 mM NACA or 0.5mM GSH (p<0.05). The addition of NAC (18.8±3.7%) or NACA (21.2±3.9%) did not improve development competence to morula and blastocysts than control (24.4±4.1%) and GSH (26.5±5.0%) (p>0.05). Our study showed that medium supplementation with CYS during IVM and IVC improved the rate of bovine embryo development but not with NAC, NACA and GSH addition.

This study examined the acute toxicity of Vital-Shoot containing reduced glutathione in male and female ICR mice. Mice were intraperitoneally injected the drug at dose levels of 0, 250, 500, 1,000 and 2,000 mg/kg body weight (BW) for single-dose toxicity test. There were no statistical differences in BW changes between the control and all treated groups. Based on hematological and blood biochemical analyses, the drug did not affect all parameters. In addition, markers for liver and kidney functions did not meaningfully change in all treated groups. Since there were no adverse effects from the drug in a single-dose intraperitoneal toxicity test, it was concluded that the lethal dose 50 (LD50) of Vital-Shoot is estimated to be >2,000 mg/kg BW.

카드뮴과 같은 중금속은 독성이 높아 수서 생물과 인간에게 해로운 영향을 미친다고 알려져 있다. 본 연구에서는 해양 섬모충 Euplotes crassus에서 카드뮴이 해독 기전에 관여하는 ABC transporters (ABCs)와 glutathione S-transferase (GST)의 유전자 발현에 미치는 영향을 조사하였 다. 총 7개의 ABCs 유전자와 1개의 GST 유전자 일부를 클로닝하여 유전자 분석을 실시하였고, 카드뮴(0.1~1 mg/l) 노출에 따른 이들 유전자의 발현 양상을 quantitative real time RT-PCR (qRT-PCR)을 이용하여 분석하였다. 염기서열 분석과 계통 분석 결과 이들 ABCs 유전자가 ABC transporter의 특징을 가지며, ABC-B/C family에 속하는 것을 확인하였고, GST 유전자는 theta isoform과 유사한 것으로 나타났다. 카드뮴에 8시간 노출시킨 결과 ABC transporter 유전자의 경우 ABCB21 유전자를 제외하고는 대부분 농도 의존적으로 유전자 발현이 유의하게 증가하였 다. GST 유전자는 0.5 mg/l에서 가장 높은 유전자 발현 양상을 보였으며, 1 mg/l에서는 발현량 이 대조군 수준으로 감소되었다. 본 연구 결과는 E. crassus의 ABC transporter와 GST 유전자가 카드뮴에 의해 유도되는 독성에 대한 방어 기전에 참여하는 것을 의미한다.

The objective of this study was to investigate to influence of glutathione (GSH) on development and antioxidant enzyme activity in tetraploid porcine embryos. Tetraploid embryos were produced using parthenogenetic 2-cell embryo by electrofusion method. Tetraploid embryo development was observed every 24 hours and intracellular antioxidant enzyme activity was measured at 120 hours after electrofusion. The 4-cell to 16-cell stage tetraploid embryos was increased in 100 and 500 μM GSH-treated groups compared control group at 48 hours (P < 0.05) but cleavage rates were not significantly different among the GSH treatment groups at 48, 72, 96, and 120 hours. Blastocyst formation was significantly increased by 300 and 500 μM GSH at 120 hours in tetraploid embryos (P < 0.05). But blastocyst cell number were not significantly different among the GSH treatment groups (16.4 ± 0.8, 16.8 ± 2.6, 18.5 ± 2.8 and 17.5 ± 1.8). The intracellular antioxidant enzyme level was increased in 500 μM GSH compared to 0 and 100 μM GSH (P < 0.05). We suggest that GSH may be improve development of tetraploid embryo in pigs.

We investigate the effect of L-glutathione (GSH), an antioxidant, treatment during the somatic cell nuclear transfer (SCNT) procedures on the in vitro development and DNA methylation status of bovine SCNT embryos. Bovine in vitro matured (IVM) oocytes were enucleated and electrofused with a donor cell, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine. The recipient oocytes or reconstituted oocytes were treated with 50 μM GSH during these SCNT procedures from enucleation to activation treatment. The SCNT embryos were cultured for 7 days to evaluate the in vitro development, apoptosis and DNA methylation in blastocysts. The apoptosis was measured by TUNEL assay and caspase-3 activity assay. Methylated DNA of SCNT embryos at the blastocyst stages was detected using a 5-methylcytidine (5-MeC) antibody. The developmental rate to the blastocyst stage was significantly higher (P<0.05) in GSH treatment group (32.5±1.2%, 78/235) than that of non-treated control SCNT embryos (22.3±1.8%, 50/224). TUNEL assay revealed that the numbers of apoptotic cells in GSH treatment group (2.3±0.4%) were significantly lower (P<0.05) than that of control (3.8±0.6%). Relative caspase-3 activity of GSH treated group was 0.8±0.06 fold compared to that of control. DNA methylation status of blastocysts in GSH treatment group (13.1±0.5, pixels/ embryo) was significantly lower (P<0.05) than that of control (17.4±0.9, pixels/embryo). These results suggest that antioxidant GSH treatment during SCNT procedures can improve the embryonic development and reduce the apoptosis and DNA methylation level of bovine SCNT embryos, which may enhance the nuclear reprogramming of bovine SCNT embryos.

Vitellogenins (Vgs) are precursors of the major egg storage protein, vitellin (Vn), in many oviparous animals. Insects Vgs are large molecules (200-kD) synthesized in the fat body in a process that involves substantial structural modifications (e.g., glycosylation, lipidation, phosphorylation, and proteolytic cleavage, etc.) of the nascent protein prior to its secretion and transport to the ovaries. However, the extent to which Vgs are processed in the fat body varies greatly among different insect groups. We were cloned Vgs partial genes PaVgs and BgVgs from Periplaneta americana and Blattella germanica. Real-time quantitative PCR shows that PaVgs and BgVgs were differential-regulated with aging. In insects, glutathione S-transferases (GSTs) are enzymes involved in detoxification of insecticides. We were cloned GST partial genes PaGST and BgGST from Periplaneta americana and Blattella germanica. Real-time quantitative PCR shows that PaGST and BgGST were up-regulated with aging, and the mRNA level of PaGST and BgGST was higher in 4℃ and 37℃ than room temperature. The expression level of PaGST and BgGST exposure to temperature stress suggests that PaGST and BgGST are up-regulated after exposure low and hige temperature treatments.

본 연구는 시험생물로서 말똥성게 (Hemicentrotus pulcherrimus) 를 이용하여 내분비계장애물질인 bisphenol A (BPA)의 독성 및 시험생물로서의 적합성 등을 조사하였 다. 말똥성게(H. pulcherrimus)의 수정 및 정상 배아발생 에 미치는 BPA의 독성을 보기 위하여 농도(0, 300, 500, 800, 1000, 1500 ppb)에서 조사하였다. BPA 노출 시 수정 률은 시험구간 내의 BPA 처리농도와 관계없이 유의적인 변화가 없었다. 정상 배아발생률은 BPA 농도가 높을수 록 유의적인 감소를 나타냈으며, 800 ppb 농도부터 유의 적인 감소를 보였다. 정상배아발생에 대한 독성값은 반 수영향농도 (EC50) 1056.1 ppb, 95% Cl 981.8~1163.9 ppb 로 나타났다. 또한 무영향농도 (NOEC)와 최소영향농도 (LOEC)는 500 ppb 및 800 ppb로 나타났다. BPA에 노출 된 배아는 농도가 증가함에 따라 발생이 정체되는 현상 이 나타났다. BPA에 노출된 pluteus 유생을 이용한 glutathione- S-transferase (GST) 유전자의 발현을 비교해본 결과 GST 유전자의 발현은 농도가 증가함에 따라 발현 이 증가하였다. 본 연구 결과, 말똥성게 (H. pulcherrimus)의 초기 배아 발생 과정 중 800 ppb 이상에서 독성을 나타냈으며 GST 유전자는 BPA 노출에 따른 위해성 평가에 생체지표유 전자로 유용하게 이용될 수 있다고 생각된다.

70% 에탄올로 양파를 추출한 다음 감압하에 농축하였고, 농축된 양파 추출물을 증류수에 재 용해시킨 용액의 pH는 5.34인 약산성으로 나타났다. 그리고 이 용액을 0.1 N NaOH 로 적정한 다음 TLC(전개용매: ethyl acetate)에서의 분리는 적 정 전보다 chromatogram의 이동 및 분리가 뛰어난 것으로 나 타났고, GST 활성 저해에도 유의성이 있는 것으로 나타났다. Silica-gel column chromatography에서 0.1 N NaOH 적정용액의 분리는 ethyl acetate(분획 A), methanol(분획 B), 50% methanol (분획 C)로 용출하였고, 용출된 각 분획은 감압하에 농축하여 GST 활성 저해 실험을 하였다. 세 부분 모두 GST 활성 저해에 유의성이 있는 것으로 나타났다. 각 분획은 GC-MS spectrometer 분석에서 두 종류 이상이 함유되어 있지만, 구성 물질은 서로 상이함을 보였고, 이들 물질의 분리와 구조 규명이 필요한 것 으로 사료된다.