• 한국미생물·생명공학회지
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• 제19권6호
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• pp.575-582
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• 1991

글루타치온 생산증대를 도모하기 위하여 글루타치온 합성에 관여하는 효소들인 GSH-I 및 GSH-II 유전자들 pBR322 벡터에 클로닝하였다. gshI 유전자를 클로닝하기 위하여 GSH-I 효소활성이 결여된 GS903 균주를 분리하였다. E. coli K-12 염색체 DNA로부터 분리된 3.6Kb PstI DNA 절편을 pBR322 벡터에 클로닝하였다. gshII 유전자는 2.2Kb PstI-BamHI DNA 절편내에 존재하며 이 절편을 pUC13 벡터에 클로닝하였다. 플라스미드의 copy number와 gsh 유전자들을 포함하고 있는 삽입 DNA의 크기의 차이에 의한 gsh 유전자들의 발현 정도를 조사하기 위하여 pGH1090, pGH101, pGH200, pGH201, pLF4, pLF6 그리고 pGH300같은 여러 플라스미드를 사용함으로써 gshI 유전자의 발현이 의해 2배이상 증가하였다. 그러나 벡터 플라스미드내에 존재하는 gsh 유전자들을 포함하는 삽입 DNA의 크기의 차이에 의해서는 gsh 유전자들의 발현이 영향을 받지 않았다.

• 약학회지
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• 제53권6호
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• pp.314-320
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• 2009

We examined metabolic conversion of cysteine into glutathione (GSH) and taurine in rat liver under oxidative stress. Administration of tert-butylhydroperoxide (t-BHP) into the portal vein of male rats resulted in a rapid elevation of serum sorbitol dehydrogenase, alanine aminotransferase, and aspartate aminotransferase activities, which decreased gradually in 24 hr. Hepatic cysteine concentration was reduced in 3 hr, and recovered progressively, reaching a level greater than 200% of the normal value in 24 hr. GSH was increased both in liver and blood at 9 hr after t-BHP challenge, whereas hypotaurine or taurine was not altered. $\gamma$-Glutamylcysteine synthetase (GCS) activity was increased from 9 hr after t-BHP treatment, but protein expression of the GCS-heavy subunit was not changed in liver. Activity or expression of cysteine dioxygenase was not affected by t-BHP treatment. Taken together, these data show that an acute oxidant challenge to the rats may induce upregulation of cysteine availability and GCS activity, resulting in an enhancement of hepatic GSH synthesis, but the increased cysteine level does not stimulate taurine synthesis via cysteine sulfinate pathway. It is indicated that the regulation of GSH and taurine biosynthesis from cysteine is not solely dependent on the cysteine concentration in rat liver under oxidative stress.

• Molecules and Cells
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• 제19권1호
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• pp.131-136
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• 2005

The ETV6-NTRK3 gene fusion, first identified in the chromosomal translocation in congenital fibrosarcoma, encodes a chimeric protein tyrosine kinase with potent transforming activity. ETV6-NTRK3-dependent transformation involves the joint action of NTRK3 signaling pathways, and aberrant cell cycle progression resulting from activation of Mek1 and Akt. The level of glutathione (GSH) was found to be markedly increased in ETV6-NTRK3-transformed NIH3T3 cells. The activities of the two GSH biosynthetic enzymes as well as of glutathione peroxidase, together with their mRNAs, were also higher in the transformed cells. The transformed cells were able to grow in the presence of GSH-depleting agents, whereas the control cells were not. L-Buthionine-(S,R)-sulfoximine (BSO) inhibited activation of Mek1 and Akt in the transformed NIH3T3 cells. These observations imply that up-regulation of GSH biosynthesis plays a central role in ETV6-NTRK3-induced transformation.

• Toxicological Research
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• 제3권2호
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• pp.129-141
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• 1987

Hepatocytes isolated from rats which have been pretreated with phenobarbital (80 mg/kg for 3 days), were able to take up N-acetylcysteine from surrounding medium and were able to synthesize the reduced glutathione ($GSH^{\ast}-3$) intracellularly. The N-acetylcysteine is quickly deacetylated after the uptake and increases the pool size of cysteine, which was very low initially (5 nmol/$10^6$ cells). From this increased intracellular cysteine pool, GSH was synthesized. Freshly isolated rat hepatocytes contained a high level of GSH (30 nmol/$10^6$ cells), but upon incubation with the diethylmaleate, it was markedly decreased (10 nmol/$10^6$ cells). The hepatocytes with depleted GSH have lost viability upon incubations with acetaminophen (5mM) and paraquat (2 mM). However, when the N-acetylcysteine (1 mM) was added to this incubation condition, these chemical induced hepatocellular necrosis were prevented for longer durations. This N-acetylcysteine dependent protective effect against the hepatotoxic chemicals was lost by adding methionine sulfoximine (10 mM), an inhibitor of GSH biosynthesis. Both the carbontetrachloride (5 mM) and chioroform (5 mM) added to the incubation medium caused rapid losses of GSH and cell viability, even without the prior depletion of cellular GSH. However, again, if the 1mM N-acetylcysteine was supplemented, the rates of losses of GSH and cell viability were retarded in both cases. Even though large amounts of the added N-acetylcysteine was present in the cell, N-acetylcysteine conjugate of acetaminophen was not formed. Instead, only large amounts of GSH conjugate of the drug was produced. Thus, it is concluded that the added N-acetylcysteine is taken up and utilized for resynthesis of GSH. In turn, this resynthesized GSH contributes to the protection against cytotoxicity inducible with hepatotoxic drugs.

• 생명과학회지
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• 제7권4호
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• pp.253-261
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• 1997

E. coli에서 글루타치온 생산 증가를 위해서 E. coli에서 분리한 gehI과 gshII유전자를 함유하고 있는 여러 재조합 플라스미드를 구성하여 도입하였다.pBR325 벡터에 gehI 유전자를 각각 1-3개를 포함한 재조합 플라스미드 및 gehI과 gehII 유전자를 동시에 갖는 재조합 플라스미드를 구성하였다. 계속적으로 반복된 gehI 유전자가 증폭된 E. colidml $\gamma $-gluramylcysteine synthetase의 효소활성은 삽입된 gehI 유전자의 부에 따라 증가하였다. 구성된 재조합 플라스미드를 함유한 E.coli의 글루타치온 생산능을 accetate kinase반응을 ATP재생계로 사용하여 조사한 결과 반복된 gehI 유전자를 함유한 E.coli의 글루타치온 생산능력은 삽입된 gehI 유전자의 수에 비례한여 증가하였으며, gehI 유전자의 추가적인 도입에 의해 글루타치온 생산능력은 2배 증가하였다. E.coli에서 글루타치온의 효소적 생산은 주로 \gamma $-gluramylcysteine synthetase의 효소활성에 의해 영향을 받았다. 가장 높은 글루타치온 생산능은 pGH501 (pUC8-gsh.I.II.III) 플라스미드를 갖는 균주에서 관찰되었다.

• 약학회지
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• 제43권3호
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• pp.385-390
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• 1999

Camptothecin (CPT) has been known to induce apoptosis in various cancer cell lines. To examine the intracellular apoptotic death signal initiated by CPT, we investigated the possible connection between caspase-3 activation and GSH depletion during CPT-induced apoptosis in HL-60 cells. Treatment of cells with $1{\;}{\mu}M$ CPT induced PARP cleavage accompanied by DNA fragmentation. z-VAD-fmk, a caspase-3 inhibitor, blocked the CPT-induced DNA fragmentation. Pretreatment of cells with N-acetylcysteine, a precursor of GSH biosynthesis, failed to inhibit CPT-induced PARP celavage and DNA gragmenatation. No significant changes in GSH depletion is not essential for caspase activation during CPT-induced apoptosis. We also investigated whether CPT-induced apoptosis is associated with changes of the levels of Bax and Bcl-2, two proteins involved in the control of apoptosis. Bcl-2 levels exhibited a late decrease compared with the kinetics of DNA fragmentation, whereas Bax levels increased more rapidly after CPT treatment. These results suggest that Bax plays more important role than Bcl-2 in inducing DNA fragmentation and may function upsteam of proteolytic activation of caspase-3 pathway in CPT-induced apoptosis.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 1991년도 춘계학술발표대회 논문집
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• pp.237-242
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• 1991

In order to increase the production of glutathione by maximizing the expression of recombinant gsh plasmids, two genes responsible for the biosynthesis of glutathione were cloned. A gshI gene was cloned onto pBR322 plasmid as 3.6Kb PstI DNA fragment from E. coli K-12 chromosomal DNA. Also gshII gene was cloned onto pUC13 plasmid as 2.2Kb PstI-BamHI DNA fragment. In order to improve the glutathione producing activity more efficiently, various recombinant plasmids containing tandem repeated gshI genes or both genes in various copy number onto the same vector were constructed. E. coli cells harboring pGH501 plasmid (pUC8-gshI$\cdot$I$\cdot$II) showed the highest glutathione synthesizing activity. The conditions for glutathione production with an ATP-generating system such as acetate kinase reaction of E. coli cells or glycolytic pathway of yeast cells were examined using the E. coli cells harboring the pGH501 plasmid. When the acetate kinase reaction of E. coli cells was used as an ATP generating system, 20mM of L-csteine was converted into glutathione with a yield of $100\%$.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.61-61
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• 2003

We have cloned the CGR1 gene encoding glutathione reductase (GR) which catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) from Candida albicans. The cgr1/cgr1 mutants were not viable when CaMAL2 promoter repressed the CGR1 expression. The growth of the mutants could be partially overcome by thiol compounds such as GSH, dithiothreitol, cysteine, N-acetylcysteine and GSSG. Interestingly, C. albicans with CGR1 overexpressed showed defective hyphal growth on solid medium and attenuated virulence. We have also cloned the GCS1 gene encoding ${\gamma}$-glutamylcysteine synthetase which catalyzes the first step of glutathione biosynthesis. The gcs1/gcs1 mutants were nonviable in minimal defined medium. The growth of the mutants could be resumed by supplementing with GSH, GSSG and ${\gamma}$-glutamylcysteine in the medium. The mutants had increased intracellular D-erythroascorbic acid level up to 2.25-fold when transferred to GSH-free medium. When the mutants were depleted of GSH, they showed typical markers of apoptosis. In conclusion, these results suggest that glutathione is an essential metabolite, and involved in hyphal growth, virulence and apoptosis in C. albicans.

• 대한의생명과학회지
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• 제7권4호
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• pp.173-179
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• 2001

To investigate the ability to decondense sperm head penetrated into cytoplasm of the oocytes and the relationship between this ability and the level of glutatione (GSH) in mouse oocyte at various maturing stages. The fertilizability of oocytes at various stages of maturation the decondensation of sperm nucleus and the formation of male pronucleus, were observed and the levels of GSH were measured in oocyte at same stages. Besides, the relation between fertilizability and level of GSH in oocyte cytoplasm treated with L-buthionine-S, R-sulfoxmine (L-BSO), the inbitor of biosynthesis of GSH, was determined. The decondensation of sperm head was not found in GV stage and L-BSO treated oocytes. In maturing oocytes (GVBD, MI), the decondensation was found, but the formation of male pronucleus was not. The levels of GSH in oocyte cytoplasm were measured; 2.2 pmol per oocyte in the ovulated and the matured in vitro each, 1.0 pmol in GV intact oocyte, 1.3 pmol in GVBD, and 1.5 pmol in MI phase oocyte. In L-BSO treated oocytes the levels of CSH were measured 0.08~o.09 pmol per oocyte, slightly lower than GV stage oocyte. In conclusion, GSH in oocyte is supposed to be synthesized and storaged in cytoplasm during maturation. The failure of decondensation in the cytoplasm of GV stage and L-BSO treated is suggested that GSH is an essential factor in decondensing the sperm head and that the a certain level of GSH, more than in GV oocyte cytoplasm, is required in decondensation.

• Animal Bioscience
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• 제35권2호
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• pp.166-176
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• 2022

Objective: Sperm is particularly susceptible to reactive oxygen species (ROS) stress. Glutathione (GSH) is an endogenous antioxidant that regulates sperm redox homeostasis. However, it is not clear whether boar sperm could utilize cysteine for synthesis GSH to protect sperm quality from ROS damage. Therefore, the present study was undertaken to elucidate the mechanism of how cysteine is involved in protecting boar sperm quality during liquid storage. Methods: Sperm motility, membrane integrity, lipid peroxidation, 4-hydroxyIlonenal (4-HNE) modifications, mitochondrial membrane potential, as well as the levels of ROS, GSH, and, ATP were evaluated. Moreover, the enzymes (GCLC: glutamate cysteine ligase; GSS: glutathione synthetase) that are involved in glutathione synthesis from cysteine precursor were detected by western blotting. Results: Compared to the control, addition of 1.25 mM cysteine to the liquid storage significantly increased boar sperm progressive motility, straight-line velocity, curvilinear velocity, beat-cross frequency, membrane integrity, mitochondrial membrane potential, ATP level, acrosome integrity, activities of superoxide dismutase and catalase, and GSH level, while reducing the ROS level, lipid peroxidation and 4-HNE modifications. It was also observed that the GCLC and GSS were expressed in boar sperm. Interestingly, when we used menadione to induce sperm with ROS stress, the menadione associated damages were observed to be reduced by the cysteine supplementation. Moreover, compared to the cysteine treatment, the γ-glutamylcysteine synthetase (γ-GCS) activity, GSH level, mitochondrial membrane potential, ATP level, membrane integrity and progressive motility in boar sperm were decreased by supplementing with an inhibitor of GSH synthesis, buthionine sulfoximine. Conclusion: These data suggest that boar sperm could biosynthesize the GSH from cysteine in vitro. Therefore, during storage, addition of cysteine improves boar sperm quality via enhancing the GSH synthesis to resist ROS stress.