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Plants possess intricate defense mechanisms to resist cadmium (Cd) stress, including strategies like metal exclusion, chelation, osmoprotection, and the regulation of photosynthesis, with antioxidants playing a pivotal role. The application of nitrogen (N) and phosphorus (P) fertilizers are reported to bolster these defenses against Cd stress. Several studies investigated the effects of N or P on Cd stress in non-woody plants and crops. However, the relationship between N, P application, and Cd stress resistance in valuable timber trees remains largely unexplored. This study delves into the Cd tolerance mechanisms of Phoebe zhennan, a forest tree species, under various treatments: Cd exposure alone, combined Cd stress with either N or P and Cd stress with both N and P application. Our results revealed that the P application enhanced root biomass and facilitated the translocation of essential nutrients like K, Mn, and Zn. Conversely, N application, especially under Cd stress, significantly inhibited plant growth, with marked reductions in leaf and stem biomass. Additionally, while the application of P resulted in reduced antioxidant enzyme levels, the combined application of N and P markedly amplified the activities of peroxidase by 266.36%, superoxide dismutase by 168.44%, and ascorbate peroxidase by 26.58% under Cd stress. This indicates an amplified capacity of the plant to neutralize reactive oxygen species. The combined treatment also led to effective regulation of nutrient and Cd distribution in roots, shoots, and leaves, illustrating a synergistic effect in mitigating toxic impact of N. The study also highlights a significant alteration in photosynthetic activities under different treatments. The N addition generally reduced chlorophyll content by over 50%, while P and NP treatments enhanced transpiration rates by up to 58.02%. Our findings suggest P and NP fertilization can manage Cd toxicity by facilitating antioxidant production, osmoprotectant, and root development, thus enhancing Cd tolerance processes, and providing novel strategies for managing Cd contamination in the environment. [ABSTRACT FROM AUTHOR]

Abstract Infectious bursal disease (IBD) of chickens is an acute, high-contact, lytic infectious disease caused by infectious bursal disease virus (IBDV). The attenuated inactivated vaccine produced by DF-1 cells is an effective control method, but the epidemic protection demands from the world poultry industry remain unfulfilled. To improve the IBDV vaccine production capacity and reduce the economic losses caused by IBDV in chicken, cellular metabolic engineering is performed on host cells. In this study, when analyzing the metabolomic after IBDV infection of DF-1 cells and the exogenous addition of reduced glutathione (GSH), we found that glutathione metabolism had an important role in the propagation of IBDV in DF-1 cells, and the glutathione synthetase gene (gss) could be a limiting regulator in glutathione metabolism. Therefore, three stable recombinant cell lines GSS-L, GSS-M, and GSS-H (gss gene overexpression with low, medium, and high mRNA levels) were screened. We found that the recombinant GSS-M cell line had the optimal regulatory effect with a 7.19 ± 0.93-fold increase in IBDV titer. We performed oxidative stress and redox status analysis on different recombinant cell lines, and found that the overexpression of gss gene significantly enhanced the ability of host cells to resist oxidative stress caused by IBDV infection. This study established a high-efficiency DF-1 cells system for IBDV vaccine production by regulating glutathione metabolism, and underscored the importance of moderate gene expression regulation on the virus reproduction providing a way for rational and precise cell engineering. Graphical Abstract

Hydrogen sulfide (H2S) regulates many critical processes of plants and their responses to abiotic stresses such as heavy metals. In the present study, the effect of sodium hydrogen sulfide (NaHS) as an H2S donor (0, and 50 μM) was investigated in soybean plants under Cd stress (0, and 50 μM). These experiments were done according to a factorial design based on a completely randomized design with three replications. Cd exposure reduced growth parameters and relative water content. Cd also caused a reduction in photosynthetic pigment, an increase in ion leakage and oxidative stress as indicated by lipid peroxidation and H2O2 production. These results demonstrated the toxicity of the applied Cd concentrations on soybean plants. However, foliar application of NaHS improved the shoot length and fresh weight on Cd-stressed plants. NaHS also alleviated the toxic effects of Cd on ion leakage, relative water content, and photosynthetic pigments of leaves. The activity of glutathione reductase and the expression of genes to the family of glutathione synthetase including glutathione synthetase, chloroplastic-like (LOC102663288) (GS288), glutathione synthetase, chloroplastic (LOC100778473) (GS473) and homoglutathione synthetase (Gene ID: 547,989) (HGS), were observed to be upregulated under the influence of NaHS treatment. However, the expression of glutathione synthetase, and chloroplastic-like glutathione synthetase (LOC100813419) (GS419)) were reduced by NaHS in Cd-stressed plants. These results confirm the regulatory effect of NaHS application on physiological parameters and glutathione metabolism at molecular levels on soybean plants.

Antioxidant protection is one of the key reactions of cardiomyocytes (CMCs) in response to myocardial damage of various origins. The thioredoxin interacting protein (TXNIP) is an inhibitor of thioredoxin (TXN). Over the recent few years, TXNIP has received significant attention due to its wide range of functions in energy metabolism. In the present work, we studied the features of the redox-thiol systems, in particular, the amount of TXNIP and glutathione synthetase (GS) as markers of oxidative damage to CMCs and antioxidant protection, respectively. This study was carried out on 38-week-old Wistar-Kyoto rats with insulin-dependent diabetes mellitus (DM) induced by streptozotocin, on 38- and 57-week-old hypertensive SHR rats and on a model of combined hypertension and DM (38-week-old SHR rats with DM). It was found that the amount of TXNIP increased in 57-week-old SHR rats, in diabetic rats and in SHR rats with DM. In 38-week-old SHR rats, the expression of TXNIP significantly decreased. The expression of GS was significantly higher compared with the controls in 57-week-old SHR rats, in DM rats and in the case of the combination of hypertension and DM. The obtained data show that myocardial damage caused by DM and hypertension are accompanied by the activation of oxidative stress and antioxidant protection.

Infectious bursal disease (IBD) of chickens is an acute, high-contact, lytic infectious disease caused by infectious bursal disease virus (IBDV). The attenuated inactivated vaccine produced by DF-1 cells is an effective control method, but the epidemic protection demands from the world poultry industry remain unfulfilled. To improve the IBDV vaccine production capacity and reduce the economic losses caused by IBDV in chicken, cellular metabolic engineering is performed on host cells. In this study, when analyzing the metabolomic after IBDV infection of DF-1 cells and the exogenous addition of reduced glutathione (GSH), we found that glutathione metabolism had an important role in the propagation of IBDV in DF-1 cells, and the glutathione synthetase gene (gss) could be a limiting regulator in glutathione metabolism. Therefore, three stable recombinant cell lines GSS-L, GSS-M, and GSS-H (gss gene overexpression with low, medium, and high mRNA levels) were screened. We found that the recombinant GSS-M cell line had the optimal regulatory effect with a 7.19 ± 0.93-fold increase in IBDV titer. We performed oxidative stress and redox status analysis on different recombinant cell lines, and found that the overexpression of gss gene significantly enhanced the ability of host cells to resist oxidative stress caused by IBDV infection. This study established a high-efficiency DF-1 cells system for IBDV vaccine production by regulating glutathione metabolism, and underscored the importance of moderate gene expression regulation on the virus reproduction providing a way for rational and precise cell engineering. [ABSTRACT FROM AUTHOR]

The Kelch-like ECH-associated protein 1–nuclear factor erythroid 2-related factor 2 (KEAP1–NRF2) system plays a central role in redox homeostasis and inflammation control. Oxidative stress or electrophilic compounds promote NRF2 stabilization and transcriptional activity by negatively regulating its inhibitor, KEAP1. We have previously reported that bromovalerylurea (BU), originally developed as a hypnotic, exerts anti-inflammatory effects in various inflammatory disease models. However, the molecular mechanism underlying its effect remains uncertain. Herein, we found that by real-time multicolor luciferase assay using stable luciferase red3 (SLR3) and green-emitting emerald luciferase (ELuc), BU potentiates NRF2-dependent transcription in the human hepatoblastoma cell line HepG2 cells, which lasted for more than 60 h. Further analysis revealed that BU promotes NRF2 accumulation and the transcription of its downstream cytoprotective genes in the HepG2 and the murine microglial cell line BV2. Keap1 knockdown did not further enhance NRF2 activity, suggesting that BU upregulates NRF2 by targeting KEAP1. Knockdown of Nfe2l2 in BV2 cells diminished the suppressive effects of BU on the production of pro-inflammatory mediators, like nitric oxide (NO) and its synthase NOS2, indicating the involvement of NRF2 in the anti-inflammatory effects of BU. These data collectively suggest that BU could be repurposed as a novel NRF2 activator to control inflammation and oxidative stress. [ABSTRACT FROM AUTHOR]

Objective To use hairy enhancer of split 1 (Hes1) to regulate the differentiation of liver epithelial progenitor cells (LEPCs) into cholangiocytes. Methods The vectors, pTet-on and pTRE2hyg-Hesl, were transfected into LEPCs. The expression of Hesl was induced by doxycycline (DOX) with different concentrations (0, 0.1, 1, 5, 10, 50, 100 and 500 g/mL). The expressions of Hesl, molecular markers of hepatocyte and cholangiocyte, glutathione synthetase (Gss), keratin 19 (Krt19) and hepatic nuclear factor 18 (HNF1β) in LEPCS were verified by Western blotting, RT-PCR, Real-time PCR, immunocytochemistry and immunofluorescence. Results The expression of Hesl in LEPCs transfected by pTet-on/pTRE2hyg-Hes1 was increased by 11.21 fold when induced by DOX at 10 ug/mL, which drove the LEPCS to differentiate into biliary epithelial cells. With increasing expression of Hes1, cholangiocyte markers, Krt19 and HNF1β, were significantly upregulated, while the hepatocyte marker, Gss, was obviously downregulated. Conclusion DOX at 10 µg/mL may induce a suitably up-regulated expression of Hes1 in LEPCs double-transfected by pTet-on and pTRE2hyg-Hes1, and the suitable high-expression rather than over-expression of Hesl can regulate LEPCs to differentiate into cholangiocytes. [ABSTRACT FROM AUTHOR]

In order to evaluate the effect of mycorrhizal fungi and phosphorous fertilizer on some root growth traits and activity of enzyme and nonenzyme antioxidants of durum wheat in rainfed conditions, an experiment was carried out as factorial based on a randomized complete block design with three replications at Sarableh Agricultural Research station during growing season 2018-2019. Four cultivars of durum wheat (Dehdasht, Zahab, Savarz and Saji) as the first factor and five levels of fertilizer source (control, 25 and 50 kg.ha-1 P, mycorrhizal fungi (GM), mycorrhizal fungi + 25 kg.ha-1 P) as the second factor were considered. The average comparison results of simple effects indicated that Zahab and Saji cultivars among cultivars and combination treatment of mycorrhizal + 25 kg / ha P and then 50 kg / ha P had the greatest effect on improving the studied traits. The interaction effect of cultivars and fertilizer sources revealed that the combined use of phosphorous fertilizer and mycorrhizal fungi had better results compared to their use alone. So that, the highest fresh and dry weight, length density, and root water content and activity of ascorbate peroxidase, peroxidase, catalase, superoxide dismutase and glutathione synthetase was obtained in combined treatment of mycorrhizal and phosphorus fertilizer at Zahab and Saji cultivars. However, the lowest specific root length, and malondialdehyde and hydrogen peroxide activity were obtained in the same treatment and cultivars. In general, the results showed that inoculation with mycorrhizal fungi in rainfed conditions, especially in Zahab and Saji cultivars, in addition to reducing the application of phosphorus fertilizer can improve root growth characteristics and activity of enzyme and nonenzyme antioxidants. As a result, it reduces the peroxidation of membrane lipids (the decline in production of malondialdehyde and hydrogen peroxide) and increases drought stress tolerance.

Antioxidant protection is one of the key reactions of cardiomyocytes (CMCs) in response to myocardial damage of various origins. The thioredoxin interacting protein (TXNIP) is an inhibitor of thioredoxin (TXN). Over the recent few years, TXNIP has received significant attention due to its wide range of functions in energy metabolism. In the present work, we studied the features of the redox-thiol systems, in particular, the amount of TXNIP and glutathione synthetase (GS) as markers of oxidative damage to CMCs and antioxidant protection, respectively. This study was carried out on 38-week-old Wistar-Kyoto rats with insulin-dependent diabetes mellitus (DM) induced by streptozotocin, on 38- and 57-week-old hypertensive SHR rats and on a model of combined hypertension and DM (38-week-old SHR rats with DM). It was found that the amount of TXNIP increased in 57-week-old SHR rats, in diabetic rats and in SHR rats with DM. In 38-week-old SHR rats, the expression of TXNIP significantly decreased. The expression of GS was significantly higher compared with the controls in 57-week-old SHR rats, in DM rats and in the case of the combination of hypertension and DM. The obtained data show that myocardial damage caused by DM and hypertension are accompanied by the activation of oxidative stress and antioxidant protection. [ABSTRACT FROM AUTHOR]

In order to evaluate the effect of mycorrhizal fungi and phosphorous fertilizer on some root growth traits and activity of enzyme and nonenzyme antioxidants of durum wheat in rainfed conditions, an experiment was carried out as factorial based on a randomized complete block design with three replications at Sarableh Agricultural Research station during growing season 2018-2019. Four cultivars of durum wheat (Dehdasht, Zahab, Savarz and Saji) as the first factor and five levels of fertilizer source (control, 25 and 50 kg.ha-1 P, mycorrhizal fungi (GM), mycorrhizal fungi + 25 kg.ha-1 P) as the second factor were considered. The average comparison results of simple effects indicated that Zahab and Saji cultivars among cultivars and combination treatment of mycorrhizal + 25 kg / ha P and then 50 kg / ha P had the greatest effect on improving the studied traits. The interaction effect of cultivars and fertilizer sources revealed that the combined use of phosphorous fertilizer and mycorrhizal fungi had better results compared to their use alone. So that, the highest fresh and dry weight, length density, and root water content and activity of ascorbate peroxidase, peroxidase, catalase, superoxide dismutase and glutathione synthetase was obtained in combined treatment of mycorrhizal and phosphorus fertilizer at Zahab and Saji cultivars. However, the lowest specific root length, and malondialdehyde and hydrogen peroxide activity were obtained in the same treatment and cultivars. In general, the results showed that inoculation with mycorrhizal fungi in rainfed conditions, especially in Zahab and Saji cultivars, in addition to reducing the application of phosphorus fertilizer can improve root growth characteristics and activity of enzyme and nonenzyme antioxidants. As a result, it reduces the peroxidation of membrane lipids (the decline in production of malondialdehyde and hydrogen peroxide) and increases drought stress tolerance. [ABSTRACT FROM AUTHOR]

Ferroptosis is a non-apoptotic form of cell death driven by iron-dependent lipid peroxidation. Recent evidence indicates that inhibiting ferroptosis could alleviate cerebral ischemia/reperfusion (CIR) injury. γ-glutamylcysteine (γ-GC), an intermediate of glutathione (GSH) synthesis, can upregulate GSH in brains. GSH is the co-factor of glutathione peroxidase 4 (GPX4), which is the negative regulator of ferroptosis. In this study, we explored the effect of γ-GC on CIR-induced neuronal ferroptosis and brain injury. We found that γ-GC significantly reduced the volume of cerebral infarction, decreased the loss of neurons and alleviated neurological dysfunction induced by CIR in rats. Further observation showed that γ-GC inhibited the CIR-caused rupture of the neuronal mitochondrial outer membrane and the disappearance of cristae, and decreased Fe2+ deposition and lipid peroxidation in rat cerebral cortices. Meanwhile, γ-GC altered the expression of some ferroptosis-related proteins in rat brains. Mechanistically, γ-GC increased the expression of GSH synthetase (GSS) for GSH synthesis via protein kinase C (PKC)ε-mediated activation of nuclear factor erythroid 2-related factor (Nrf2). Our findings suggest that γ-GC not only serves as a raw material but also increases the GSS expression for GSH synthesis against CIR-induced lipid peroxidation and ferroptosis. Our study strongly suggests that γ-GC has potential for treating CIR injury. [ABSTRACT FROM AUTHOR]

Glutathione (GSH) is synthesized in two ATP‐dependent reactions by glutamate‐cysteine ligase (Gcl) and glutathione synthetase (Gs). Myxococcus xanthus, a gram‐negative bacterium belonging to δ‐proteobacteria, possesses mxGcl and mxGs, which have high sequence identity with the enzymes from plants and bacteria, respectively. MxGcl2 was activated by Mn2+, but not by Mg2+, and stabilized in the presence of 5 mM Mn2+ or Mg2+. Sequence comparison of mxGcl2 and Brassica juncea Gcl indicated that they have the same active site residues, except for Tyr330, which interacts with Cys and which in mxGcl2 is represented by Leu267. The substitution of Leu267 with Tyr resulted in the loss of mxGcl2 activity, but that with Met (found in cyanobacterial Gcls) increased the mxGcl2 affinity for Cys. GSH and its oxidized form GSSG equally inhibited the activity of mxGcl2; the inhibition was augmented by ATP at concentrations >3 mM. Buthionine sulfoximine inactivated mxGcl2 with Ki = 2.1 μM, which was lower than those for Gcls from other organisms. The mxGcl2 activity was also suppressed by pyrophosphate and polyphosphates. MxGs was a dimer, and its activity was induced by Mg2+ but strongly inhibited by Mn2+ even in the presence of 10 mM Mg2+. MxGs was inhibited by GSSG at Ki = 3.6 mM. Approximately 1 mM GSH was generated with 3 units of mxGcl2 and 6 units of mxGs from 5 mM Glu, Cys, and Gly, and 10 mM ATP. Our results suggest that GSH production in M. xanthus mostly depends on mxGcl2 activity. [ABSTRACT FROM AUTHOR]

[Display omitted] • Furfural was found as the key inhibitor in lignocellulosic hydrolysates that affects the cell growth and SA production of Y. lipolytica engineered strains. • YlGsh2 overexpression improved furfural detoxification of Y. lipolytica through increasing intracellular glutathione content. • Engineered Hi-SA2-YlGsh2 strain produced 45.34 g/L SA with a yield of 0.71 g/g in fed-batch fermentation using undetoxified corncob hydrolysate. • This study demonstrated the potential of Y. lipolytica engineered strains to efficiently produce bio-based SA from toxic lignocellulosic biomass at low pH. Succinic acid (SA) is a valuable C4 platform chemical with diverse applications. Lignocellulosic biomass represents an abundant and renewable carbon resource for microbial production of SA. However, the presence of toxic compounds in pretreated lignocellulosic hydrolysates poses challenges to cell metabolism, leading to inefficient SA production. Here, engineered Yarrowia lipolytica Hi-SA2 was shown to utilize glucose and xylose from corncob hydrolysate to produce 32.6 g/L SA in shaking flasks. The high concentration of undetoxified hydrolysates significantly inhibited yeast growth and SA biosynthesis, with furfural identified as the key inhibitor. Through overexpressing glutathione synthetase encoding gene YlGsh2 , the tolerance of engineered strain to furfural and toxic hydrolysate was significantly improved. In a 5-L bioreactor, Hi-SA2-YlGsh2 strain produced 45.34 g/L SA within 32 h, with a final pH of 3.28. This study provides a sustainable process for bio-based SA production, highlighting the efficient SA synthesis from lignocellulosic biomass through low pH fermentation. [ABSTRACT FROM AUTHOR]

P2X7 receptor (P2X7R) regulates inducible nitric oxide synthase (iNOS) expression/activity in response to various harmful insults. Since P2X7R deletion paradoxically decreases the basal glutathione (GSH) level in the mouse hippocampus, it is likely that P2X7R may increase the demand for GSH for the maintenance of the intracellular redox state or affect other antioxidant defense systems. Therefore, the present study was designed to elucidate whether P2X7R affects nuclear factor-erythroid 2-related factor 2 (Nrf2) activity/expression and GSH synthesis under nitrosative stress in response to lipopolysaccharide (LPS)-induced neuroinflammation. In the present study, P2X7R deletion attenuated iNOS upregulation and Nrf2 degradation induced by LPS. Compatible with iNOS induction, P2X7R deletion decreased S-nitrosylated (SNO)-cysteine production under physiological and post-LPS treated conditions. P2X7R deletion also ameliorated the decreases in GSH, glutathione synthetase, GS and ASCT2 levels concomitant with the reduced S-nitrosylations of GS and ASCT2 following LPS treatment. Furthermore, LPS upregulated cystine:glutamate transporter (xCT) and glutaminase in P2X7R+/+ mice, which were abrogated by P2X7R deletion. LPS did not affect GCLC level in both P2X7R+/+ and P2X7R−/− mice. Therefore, our findings indicate that P2X7R may augment LPS-induced neuroinflammation by leading to Nrf2 degradation, aberrant glutamate-glutamine cycle and impaired cystine/cysteine uptake, which would inhibit GSH biosynthesis. Therefore, we suggest that the targeting of P2X7R, which would exert nitrosative stress with iNOS in a positive feedback manner, may be one of the important therapeutic strategies of nitrosative stress under pathophysiological conditions. [ABSTRACT FROM AUTHOR]

In this study, the roles of glutathione (GSH), homoglutathione (hGSH), and their ratio in symbiotic nodule development and functioning, as well as in defense responses accompanying ineffective nodulation in pea (Pisum sativum) were investigated. The expression of genes involved in (h)GSH biosynthesis, thiol content, and localization of the reduced form of GSH were analyzed in nodules of wild-type pea plants and mutants sym33-3 (weak allele, "locked" infection threads, occasional bacterial release, and defense reactions) and sym33-2 (strong allele, "locked" infection threads, defense reactions), and sym40-1 (abnormal bacteroids, oxidative stress, early senescence, and defense reactions). The effects of (h)GSH depletion and GSH treatment on nodule number and development were also examined. The GSH:hGSH ratio was found to be higher in nodules than in uninoculated roots in all genotypes analyzed, with the highest value being detected in wild-type nodules. Moreover, it was demonstrated, that a hGSHS -to- GSHS switch in gene expression in nodule tissue occurs only after bacterial release and leads to an increase in the GSH:hGSH ratio. Ineffective nodules showed variable GSH:hGSH ratios that correlated with the stage of nodule development. Changes in the levels of both thiols led to the activation of defense responses in nodules. The application of a (h)GSH biosynthesis inhibitor disrupted the nitrogen fixation zone in wild-type nodules, affected symbiosome formation in sym40-1 mutant nodules, and meristem functioning and infection thread growth in sym33-3 mutant nodules. An increase in the levels of both thiols following GSH treatment promoted both infection and extension of defense responses in sym33-3 nodules, whereas a similar increase in sym40-1 nodules led to the formation of infected cells resembling wild-type nitrogen-fixing cells and the disappearance of an early senescence zone in the base of the nodule. Meanwhile, an increase in hGSH levels in sym40-1 nodules resulting from GSH treatment manifested as a restriction of infection similar to that seen in untreated sym33-3 nodules. These findings indicated that a certain level of thiols is required for proper symbiotic nitrogen fixation and that changes in thiol content or the GSH:hGSH ratio are associated with different abnormalities and defense responses. [ABSTRACT FROM AUTHOR]

In order to investigate the effect of growth-promoting bacteria on physiologic traitsof new dryland wheat cultivars, a factorial field experiment was carried out in a randomized complete block design with three replications on the farms of Sarablah Agricultural Research Center in the cropping season during 2019-2020. Experimental treatments included different wheat cultivars (Sardari, Karim, Koohdasht, and Rijaw) and different fertilizer sources including control (without fertilizer source), 50% N fertilizer, Azospirillum+50% N fertilizer, Azetobacter + 50% N fertilizer, Azospirillum + Azetobacter+50% N fertilizer, and 100% N fertilizer. The results of this experiment showed that the interaction effect of cultivar × fertilizer sources had a significant effect on the activity of some antioxidant enzymes and physiological properties. Rijaw cultivar × Azospirillum + Azetobacter+ 50% N fertilizer increased the activities of ascorbate peroxidase (80.9%), glutathione peroxidase (80%), catalase (71.7%), peroxidase (74.1%), superoxide dismutase (74%), carotenoids (72.1%), chlorophyll a (86.1%), chlorophyll b (88.2%), and relative water content (45.4%) while reduced the amount of Malondialdehyde (65.9%) and hydrogen peroxide (91.8%). Moreover, Sardari cultivar × control treatment (without fertilizer sources) had the lowest activities of ascorbate peroxidase, peroxidase, superoxide dismutase, and photosynthetic pigments. Therefore, according to the results of the study, wheat cultivar Rijaw × Azospirillum + Azetobacter+ 50% N fertilizer may be recommended for cultivation in dryland conditions considering the improved physiological characteristics and ultimately increased plant growth.

The aim of this study was to investigate the effects of low temperature (4 °C), cadmium sulphate (100 μM), or their combination on content of free proline (Pro), glutathione (GSH), and phytochelatins (PCs) in wheat (Triticum aestivum L.) leaves. Results revealed an increase in proline and phytochelatins accumulation in leaves of wheat seedlings along with enhanced cold tolerance at the low temperature, CdSO4, and their combination. Moreover, there were increases in mRNA content of TaP5CS and TaPCS1 genes, encoding ∆1-pyrroline-5-carboxylate synthase (P5CS) and phytochelatin synthases (PCS), respectively. A rapid increase in glutathione content was found within 1 h of exposure to the low temperature and its combination with CdSO4, followed by a drop. However, upregulation of the TaGS1 gene, encoding glutathione synthetase (GS), was maintained during 7 d. A significant decrease in glutathione content on the seventh day of exposure to the low temperature, CdSO4, and their combination was most probably due to its involvement in cadmium detoxification and/or in phytochelatin synthesis. Our data suggest that proline, glutathione, and phytochelatins may be important for plant tolerance to low temperature and cadmium stress in wheat.

Ferroptosis is a non-apoptotic form of cell death driven by iron-dependent lipid peroxidation. Recent evidence indicates that inhibiting ferroptosis could alleviate cerebral ischemia/reperfusion (CIR) injury. γ-glutamylcysteine (γ-GC), an intermediate of glutathione (GSH) synthesis, can upregulate GSH in brains. GSH is the co-factor of glutathione peroxidase 4 (GPX4), which is the negative regulator of ferroptosis. In this study, we explored the effect of γ-GC on CIR-induced neuronal ferroptosis and brain injury. We found that γ-GC significantly reduced the volume of cerebral infarction, decreased the loss of neurons and alleviated neurological dysfunction induced by CIR in rats. Further observation showed that γ-GC inhibited the CIR-caused rupture of the neuronal mitochondrial outer membrane and the disappearance of cristae, and decreased Fe2+ deposition and lipid peroxidation in rat cerebral cortices. Meanwhile, γ-GC altered the expression of some ferroptosis-related proteins in rat brains. Mechanistically, γ-GC increased the expression of GSH synthetase (GSS) for GSH synthesis via protein kinase C (PKC)ε-mediated activation of nuclear factor erythroid 2-related factor (Nrf2). Our findings suggest that γ-GC not only serves as a raw material but also increases the GSS expression for GSH synthesis against CIR-induced lipid peroxidation and ferroptosis. Our study strongly suggests that γ-GC has potential for treating CIR injury.

P2X7 receptor (P2X7R) regulates inducible nitric oxide synthase (iNOS) expression/activity in response to various harmful insults. Since P2X7R deletion paradoxically decreases the basal glutathione (GSH) level in the mouse hippocampus, it is likely that P2X7R may increase the demand for GSH for the maintenance of the intracellular redox state or affect other antioxidant defense systems. Therefore, the present study was designed to elucidate whether P2X7R affects nuclear factor-erythroid 2-related factor 2 (Nrf2) activity/expression and GSH synthesis under nitrosative stress in response to lipopolysaccharide (LPS)-induced neuroinflammation. In the present study, P2X7R deletion attenuated iNOS upregulation and Nrf2 degradation induced by LPS. Compatible with iNOS induction, P2X7R deletion decreased S-nitrosylated (SNO)-cysteine production under physiological and post-LPS treated conditions. P2X7R deletion also ameliorated the decreases in GSH, glutathione synthetase, GS and ASCT2 levels concomitant with the reduced S-nitrosylations of GS and ASCT2 following LPS treatment. Furthermore, LPS upregulated cystine:glutamate transporter (xCT) and glutaminase in P2X7R+/+ mice, which were abrogated by P2X7R deletion. LPS did not affect GCLC level in both P2X7R+/+ and P2X7R−/− mice. Therefore, our findings indicate that P2X7R may augment LPS-induced neuroinflammation by leading to Nrf2 degradation, aberrant glutamate-glutamine cycle and impaired cystine/cysteine uptake, which would inhibit GSH biosynthesis. Therefore, we suggest that the targeting of P2X7R, which would exert nitrosative stress with iNOS in a positive feedback manner, may be one of the important therapeutic strategies of nitrosative stress under pathophysiological conditions.

Glutathione synthetase (GSS) deficiency is a rare disorder, occurring with a frequency of less than 1 in 100,000 individuals worldwide. The clinical presentation may vary from mild to severe, and manifestations include hemolytic anemia, hyperbilirubinemia, metabolic acidosis, neurological problems, and sepsis. Herein, we present a case of a newborn boy with the most severe phenotype of GSS deficiency, diagnosed based on clinical features and increased urinary 5-oxoproline levels determined via gas chromatography mass spectrometry (GCMS) testing. [ABSTRACT FROM AUTHOR]

Polydatin (PD) is a bio-active ingredient with known anti-tumor effects. However, its specific protein targets yet have not been systematically screened, and the molecular anti-tumor mechanism is still unclear. Here, proteomic-chip was efficiently used to screen potential targets of PD. First, we investigated through animal experiment and proteomics studies, and found that polydatin play an important role in tumor cells. Then, the red-green fluorescent of polydatin was compared comprehensively to screen its targets on chip, followed by bioinformatics analysis. Glutathione synthetase (GSS) was selected as candidate research target. After a series of molecular biological experiments GSS was confirmed a target protein for PD in vitro. Moreover, we also found that PD can significantly inhibit the activity of GSS in vitro and live cells. Our findings reveal that PD could be a selective small-molecule GSS enzyme activity inhibitor and GSS could be a potential therapeutic target in cancer. [ABSTRACT FROM AUTHOR]

Maternal nutrition during pregnancy influences offspring health. Dietary supplementation of pregnant women with (n‐3) long‐chain polyunsaturated fatty acids (PUFA) was shown to exert beneficial effects on offspring, through yet unknown mechanisms. Here, we conducted a dietary intervention study on a cohort of 10 women diagnosed with threatened preterm labor with a nutritional integration with eicosapentaenoic and docosahexaenoic acids. Microvesicles (MV) isolated form arterial cord blood of the treated cohort offspring and also of a randomized selection of 10 untreated preterm and 12 term newborns, were characterized by dynamic light scattering and analyzed by proteomic and statistical analysis. Glutathione synthetase was the protein bearing the highest discrimination ability between cohorts. ELISA assay showed that glutathione synthetase was more abundant in cord blood from untreated preterm compared to the other conditions. Assay of free SH‐groups showed that serum of preterm subjects was oxidized. Data suggest that preterm suffer from oxidative stress, which was lower in the treated cohort. This study confirms that MV are a representative sample of the individual status and the efficacy of dietary intervention with PUFA in human pregnancy in terms of lowered inflammatory status, increased gestational age and weight at birth. [ABSTRACT FROM AUTHOR]

Plants possess intricate defense mechanisms to resist cadmium (Cd) stress, including strategies like metal exclusion, chelation, osmoprotection, and the regulation of photosynthesis, with antioxidants playing a pivotal role. The application of nitrogen (N) and phosphorus (P) fertilizers are reported to bolster these defenses against Cd stress. Several studies investigated the effects of N or P on Cd stress in non-woody plants and crops. However, the relationship between N, P application, and Cd stress resistance in valuable timber trees remains largely unexplored. This study delves into the Cd tolerance mechanisms of Phoebe zhennan , a forest tree species, under various treatments: Cd exposure alone, combined Cd stress with either N or P and Cd stress with both N and P application. Our results revealed that the P application enhanced root biomass and facilitated the translocation of essential nutrients like K, Mn, and Zn. Conversely, N application, especially under Cd stress, significantly inhibited plant growth, with marked reductions in leaf and stem biomass. Additionally, while the application of P resulted in reduced antioxidant enzyme levels, the combined application of N and P markedly amplified the activities of peroxidase by 266.36%, superoxide dismutase by 168.44%, and ascorbate peroxidase by 26.58% under Cd stress. This indicates an amplified capacity of the plant to neutralize reactive oxygen species. The combined treatment also led to effective regulation of nutrient and Cd distribution in roots, shoots, and leaves, illustrating a synergistic effect in mitigating toxic impact of N. The study also highlights a significant alteration in photosynthetic activities under different treatments. The N addition generally reduced chlorophyll content by over 50%, while P and NP treatments enhanced transpiration rates by up to 58.02%. Our findings suggest P and NP fertilization can manage Cd toxicity by facilitating antioxidant production, osmoprotectant, and root development, thus enhancing Cd tolerance processes, and providing novel strategies for managing Cd contamination in the environment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Shoaib, Lin, Mughal, Wu, Sun, Zhang and Pan.)

Glutathione synthetase deficiency (GSSD) is a rare inborn error of glutathione metabolism with autosomal recessive inheritance. The severe form of the disease is characterized by acute metabolic acidosis, usually present in the neonatal period with hemolytic anemia and progressive encephalopathy. A case of a male newborn infant who had severe metabolic acidosis with high anion gap, hemolytic anemia, and hyperbilirubinemia is reported. A high level of 5-oxoproline was detected in his urine and a diagnosis of generalized GSSD was made. DNA sequence analysis revealed the infant to be compound heterozygous with two mutations, c.738dupG in exon 8 of GSS gene resulting in p.S247fs and a repetitive sequence in exon 3 of GSS gene. Treatment after diagnosis of GSSD included supplementation with antioxidants and oral sodium hydrogen bicarbonate. However, he maintained a variable degree of metabolic acidosis and succumbed shortly after his parents requested discontinuation of therapy because of dismal prognosis and medical futility when he was 18 days old.

Glutathione is the most important part of a plant’s antioxidant defense system. Two enzymes are involved in cellular glutathione biosynthesis: glutamylcysteine ligase and glutathione synthetase, of which the latter catalyzes the transfer of glycine to the glutamylcysteine dipeptide. Transgenic plants with an elevated expression level of the glutathione synthetase genes are known to be tolerant to heavy metals. However, our knowledge on their tolerance to other types of the abiotic stress is insufficient. The goal of this study is to produce transgenic tobacco plants with a constitutive expression of the glutathione synthetase gene BnGSH from rapeseed and to estimate their growth parameters under the normal conditions, as well as under the effects of salt, drought, and cold stresses. We generate 17 lines of transgenic plants with the rapeseed BnGSH gene under the control of the 35S promoter by agrobacterium-mediated transformation. The transgene presence is confirmed by the PCR and histochemical analysis of the activity of the GUS reporter gene. Twelve lines with the highest expression level of the BnGSH gene are chosen based on the results of RT-PCR. The following morphological parameters are measured: stem height, leaf area, flower length, fresh and dry weights of shoots, and root length. Some transgenic plants demonstrated increased productivity both under normal conditions and under the effect of a high salinity stress. However, no change in the tolerance to drought and cold was observed in the transgenic plants. [ABSTRACT FROM AUTHOR]

Glutathione (GSH) fulfills a variety of metabolic functions, participates in oxidative stress response, and defends against toxic actions of heavy metals and xenobiotics. In this study, GSH was detected in Rhodosporidium diobovatum by high-performance liquid chromatography (HPLC). Then, two novel enzymes from R. diobovatum were characterized that convert glutamate, cysteine, and glycine into GSH. Based on reverse transcription PCR, we obtained the glutathione synthetase gene ( GSH2), 1866 bp, coding for a 56.6-kDa protein, and the glutamate cysteine ligase gene ( GSH1), 2469 bp, coding for a 90.5-kDa protein. The role of GSH1 and GSH2 for the biosynthesis of GSH in the marine yeast R. diobovatum was determined by deletions using the CRISPR-Cas9 nuclease system and enzymatic activity. These results also showed that GSH1 and GSH2 were involved in the production of GSH and are thus being potentially useful to engineer GSH pathways. Alternatively, pET- GSH constructed using vitro recombination could be used to detect the function of genes related to GSH biosynthesis. Finally, the fermentation parameters determined in the present study provide a reference for industrial GSH production in R. diobovatum. [ABSTRACT FROM AUTHOR]

Objective To study the toxic effects and mechanism of buthionine sulfoximine ( BSO) on human normal hepatocytes L&2 and its mechanism. Methods ① Human normal hepatocytes L&2 were incubated with 0，10，25，50, 100，and 200 #mol/L, BSO for 48 h，the cell viability was measured by MTT and typan blue staining.② L-02 cells were incubated with 0，10，25，50，100，and 200 #mol/L BSO for 48 h，the content of glutathione (GSH) was detected by DTNBmethod.③ L&2 cells were incubated with 200 #mol/L BSOfor 0，2，12，24，36，and 48 h，Wetern blotting was applied to detect the phosphorylation of p38.④ L-02 cells were incubated with 200 #mol/L BSO for 0，2，12，24，36, and 48 h，fluorescence probe H2DCFDA was used to observe the production of ROS in L-02 cells.⑤ L-02 cells were di-vided into the control group，BSO group，SB203580 group，and BSO n SB203580 group. No drugs were added to the con¬ trol group，BSO group was given 200 #mol/I, BSO，while the SB203580 group was added witli 20 #mol/L, SB203580，and the BSO + SB203580 group was first incubated with 20 #mol/L SB203580 for 15 min，then was added with 200 #mol/L BSO. After 24 h，the survival rate of each group was measured by MTT method，and the level of ROS was detected by fluo- rescencc probe H2DCFDA. Results ① BSO reduced the survival rate of L-02 cells in a dose-dependent manner，and the minimum toxic dose was 50 #mol/L (! <0. 05).② BSOcould reduce the GSH content of L-02 cells in a dose-dependent manner，and it reached the minimumvalue since the concentration of BSOwas 25 #mol/L.③ BSOincreased the phospho-rylation level of p38 in L-02 cells in a time-dependent manner (! <0. 05).④ Compared with control group，the level of ROS increased in L-02 cells when treated with BSO $ and reached its peak at 24 h.⑤ The ROS level in the BSOgroup was higher and the cell survival rate was lower than those of the control group ( both !< 0. 05 ). There was no statistically significant diference in the ROS level and cell survival rate bet^veen the control group and SB203580 group ( ! >0. 05). The ROS level in the BSO n SB203580 group was lower and the cell survival rate was higher than those of the BSOgroup ( both ! < 0. 05). Conclusion BSO has certain cytotoxicity to human normal liver cells by increasing the phosphorylation level of p38 and ROS production，and decreasing the content of GSH [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is one of the most frequent malignancies in the Western world. Early tumor detection and intervention are important determinants on CRC patient survival. During early tumor proliferation, dissemination and angiogenesis, platelets store and segregate proteins actively and selectively. Hence, the platelet proteome is a potential source of biomarkers denoting early malignancy. By comparing protein profiles of platelets between healthy volunteers ( n = 12) and patients with early- ( n = 7) and late-stage ( n = 5) CRCs using multiplex fluorescence two-dimensional gel electrophoresis (2D-DIGE), we aimed at identifying differentially regulated proteins within platelets. By inter-group comparisons, 94 differentially expressed protein spots were detected ( p < 0.05) between healthy controls and patients with early- and late-stage CRCs and revealed distinct separations between all three groups in principal component analyses. 54 proteins of interest were identified by mass spectrometry and resulted in high-ranked Ingenuity Pathway Analysis networks associated with Cellular function and maintenance, Cellular assembly and organization, Developmental disorder and Organismal injury and abnormalities ( p < 0.0001 to p = 0.0495). Target proteins were validated by multiplex fluorescence-based Western blot analyses using an additional, independent cohort of platelet protein samples [healthy controls ( n = 15), early-stage CRCs ( n = 15), late-stage CRCs ( n = 15)]. Two proteins-clusterin and glutathione synthetase (GSH-S)-featured high impact and were subsequently validated in this independent clinical cohort distinguishing healthy controls from patients with early- and late-stage CRCs. Thus, the potential of clusterin and GSH-S as platelet biomarkers for early detection of CRC could improve existing screening modalities in clinical application and should be confirmed in a prospective multicenter trial. [ABSTRACT FROM AUTHOR]

• For the first time, Glutathione synthetase from Hydra vulgaris Ind-Pune has been cloned, expressed, and characterized. • Homology modeling indicates that it is comparable to GS in higher vertebrates. • Transcripts of Hv GS were only detected in the ectoderm of whole Hydra. • Expression and enzymatic activity of Hv GS were induced by exposure to H 2 O 2. [ABSTRACT FROM AUTHOR]