The effect of glutathione on glucosinolate biosynthesis through the sulfur assimilation pathway in pakchoi associated with the growth conditions

Glucosinolates(GSLs) are a group of nitrogen-and sulfur-containing secondary metabolites, synthesized primarily in members of the Brassicaceae family, that play an important role in food flavor, plant antimicrobial activity, resistance to insect attack, stress tolerance, and human anti-cancer effects. As a sulfur-containing compound, glutathione has a strong connection with GSLs biosynthesis as a sulfur donor or redox system, and exists in reduced(glutathione;GSH) and oxidized(glutathione disulfide;GSSG) forms. However, the mechanism of GSH regulating GSLs biosynthesis remainds unclear. Hence, the exogenous therapy to pakchoi under normal growth condition and sulfur deficiency condition were conducted in this work to explore the relevant mechanism. The results showed that exogenous application of buthionine sulfoximine, an inhibitor of GSH synthesis, decreased the transcript levels of GSLs synthesis-related genes and transcription factors, as well as sulfur assimilation-related genes under the normal growth condition. Application of exogenous GSH inhibited the expression of GSLs synthesis-and sulfur assimilation-related genes under the normal condition, while the GSLs biosynthesis and the sulfur assimilation pathway were activated by exogenous application of GSH when the content of GSH in vivo of plants decreased owing to sulfur deficiency. Moreover,exogenous application of GSSG increased the transcript levels of GSLs synthesis-and sulfur assimilation-related genes under the normal growth condition and under sulfur deficiency. The present work provides new insights into the molecular mechanisms of GSLs biosynthesis underlying glutathione regulation.

Silent information regulator sirtuin 1 ameliorates acute liver failure via the p53/glutathione peroxidase 4/gasdermin D axis

BACKGROUND Acute liver failure(ALF)has a high mortality with widespread hepatocyte death involving ferroptosis and pyroptosis.The silent information regulator sirtuin 1(SIRT1)-mediated deacetylation affects multiple biological processes,including cellular senescence,apoptosis,sugar and lipid metabolism,oxidative stress,and inflammation.AIM To investigate the association between ferroptosis and pyroptosis and the upstream regulatory mechanisms.METHODS This study included 30 patients with ALF and 30 healthy individuals who underwent serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)testing.C57BL/6 mice were also intraperitoneally pretreated with SIRT1,p53,or glutathione peroxidase 4(GPX4)inducers and inhibitors and injected with lipopolysaccharide(LPS)/D-galactosamine(D-GalN)to induce ALF.Gasdermin D(GSDMD)^(-/-)mice were used as an experimental group.Histological changes in liver tissue were monitored by hematoxylin and eosin staining.ALT,AST,glutathione,reactive oxygen species,and iron levels were measured using commercial kits.Ferroptosis-and pyroptosis-related protein and mRNA expression was detected by western blot and quantitative real-time polymerase chain reaction.SIRT1,p53,and GSDMD were assessed by immunofluorescence analysis.RESULTS Serum AST and ALT levels were elevated in patients with ALF.SIRT1,solute carrier family 7a member 11(SLC7A11),and GPX4 protein expression was decreased and acetylated p5,p53,GSDMD,and acyl-CoA synthetase long-chain family member 4(ACSL4)protein levels were elevated in human ALF liver tissue.In the p53 and ferroptosis inhibitor-treated and GSDMD^(-/-)groups,serum interleukin(IL)-1β,tumour necrosis factor alpha,IL-6,IL-2 and C-C motif ligand 2 levels were decreased and hepatic impairment was mitigated.In mice with GSDMD knockout,p53 was reduced,GPX4 was increased,and ferroptotic events(depletion of SLC7A11,elevation of ACSL4,and iron accumulation)were detected.In vitro,knockdown of p53 and overexpression of GPX4 reduced AST and ALT levels,the cytostatic rate,and GSDMD expression,restoring SLC7A11 depletion.Moreover,SIRT1 agonist and overexpression of SIRT1 alleviated acute liver injury and decreased iron deposition compared with results in the model group,accompanied by reduced p53,GSDMD,and ACSL4,and increased SLC7A11 and GPX4.Inactivation of SIRT1 exacerbated ferroptotic and pyroptotic cell death and aggravated liver injury in LPS/D-GalNinduced in vitro and in vivo models.CONCLUSION SIRT1 activation attenuates LPS/D-GalN-induced ferroptosis and pyroptosis by inhibiting the p53/GPX4/GSDMD signaling pathway in ALF.

Influence of temperature on glutathione level and glutathione-related enzyme activities of Antarctic ice microalgae Chlamydomonas sp.ICE-L

GSH system plays a role in the control of the redox balance state, anti-oxidation and protecting life from injury of ROS （ reactive oxygen species）. In present paper, the possible GSH system of Chlamydomonas sp. ICE-L has been investigated by evaluating GSH and GSH-related enzymatic responses at different temperatures using spectrophotometer methods. The results showed that the GSH system is correlated positively to low temperature, and other factors but GR are correlated negatively to high temperature. So GSH and GSH-related enzymes play an important role in the adaptation of Antarctic ice microalgae to low temperature.

DMMIC derivatization-assisted liquid chromatography-mass spectrometry method for metabolite profiling of the glutathione anabolic pathway in esophageal cancer tissues and cells

In this work,a new pyrylium derivatization-assisted liquid chromatography-mass spectrometry(LC-MS)method was developed for metabolite profiling of the glutathione anabolic pathway(GAP)in cancer tissues and cells.The pyrylium salt of 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate(DMMIC)was used to label the amino group of metabolites,and a reductant of dithiothreitol(DTT)was employed to stabilize the thiol group.By combining DMMIC derivatization with LC-MS,it was feasible to quantify the 13 main metabolites on the GAP in complex biological samples,which had good linearity(R^(2)=0.99810.9999),precision(interday precision of 1.6%e19.0%and intraday precision of 1.4%e19.8%)and accuracy(83.4%-115.7%).Moreover,the recovery assessments in tissues(82.5%e107.3%)and in cells(98.1%e118.9%)with GSH-^(13)C2,^(15)N,and Cys-^(15)N demonstrated the reliability of the method in detecting tissues and cells.Following a methodological evaluation,the method was applied successfully to investigate difference in the GAP between the carcinoma and para-carcinoma tissues of esophageal squamous cell carcinoma(ESCC)and the effect of p-hydroxycinnamaldehyde(CMSP)on the GAP in KYSE150 esophageal cancer cells.The results demonstrate that the developed method provides a promising new tool to elucidate the roles of GAP in physiological and pathological processes,which can contribute to research on drugs and diseases.

Glutathione S-Transferase(GST)Identified from Giant Kelp Macrocystis pyrifera Increases the Copper Tolerance of Synechococcus elongatus PCC 7942

The glutathione S-transferases gene family plays an important regulatory role in growth and development,and responses to environmental change.In this study,six complete GST genes(Mp GST1,Mp GST2,Mp GST3,MpGST4,Mp GST5,and Mp GST6)were cloned from the gametophytes of brown alga Macrocystis pyrifera.Subsequent bioinformatics analysis showed that these six genes encoded proteins with 202,216,288,201,205,and 201 aa,respectively.Moreover,Mp GST3 differs from the other GST genes.Phylogenetic analysis suggested that MpGST3 belongs to the Ure2p type GST.Domain analysis suggested that the other GSTs from M.pyrifera belong to the soluble GST family and form an independent branch with the GSTs found in the other macroalgae,suggesting that a new GST type was formed during macroalgal evolution.GST genes were upregulated in M.pyrifera when 2.5 mg L^(-1)Cu ions were added to the medium.Six GST genes were integrated into the genome of Synechococcus elongatus PCC 7942,and their functions were verified by measuring light absorbance,photosynthetic pigment content,and photosynthetic parameters of the transformed strains under 0.3 mg L^(-1)Cu ion stress.The results showed much higher levels of various parameters in the transformed strains than in the wild strain.The transformed strains(with the MpGST genes)showed significantly enhanced resistance to Cu ion stress,while the wild strain almost died.The results of this study lay a theoretical foundation for further research on the Cu ion stress resistance function of GSTs in M.pyrifera.

Molecular identification and biochemical characteristics of a delta class glutathione S-transferase gene(FcδGST)from Chinese shrimp Fenneropenaeus chinensis

Glutathione S-transferases(GSTs)are a superfamily of multifunction enzymes involved in the regulation of redox homeostasis and innate immune responses against various pathogenic infections in marine invertebrates.In the present study,a delta class GST gene(designated as FcδGST)was cloned from Fenneropenaeus chinensis using rapid amplification of c DNA ends(RACE)technology.The complete cDNA sequence of FcδGST was 780 bp in length,which includes a 27-bp 5′non-coding region(UTR),a 117-bp 3′UTR,a 636-bp open reading frame(ORF),and a polyadenylate signal site(AATAAA)presented at the upstream of poly A tail.The FcδGST gene encoded 211 amino acids peptide,including a GST_N domain and a GST_C domain,and exhibited high similarity with previously reported delta GSTs.The predicted molecular mass of FcδGST protein was 23.39 kDa,and its theoretical isoelectric point(pI)was 5.34.The FcδGST mRNA transcripts were ubiquitously expressed in all the tested tissues,with the highest expression level in hemocytes and hepatopancreas.During the stimulation of Vibrio anguillarum or white spot syndrome virus(WSSV),the m RNA expression of FcδGST in hemocytes and hepatopancreas revealed significant up-regulation.The purified recombinant FcδGST protein(designated as rFcδGST)exhibited specific catalytic activity against 1-chloro-2,4-dinitrobenzene(CDNB)substrate with relatively low stable enzymatic activities.These results indicated that FcδGST was a fragile but typical novel delta class GST member and potentially involved in the innate immune responses of F.chinensis.

N-acetylcysteine and reduced glutathione reverse flupirtine-induced liver injury and pro⁃duce other beneficial effects in combination with flupirtine

OBJECTIVE To assess whether N-acetylcysteine(NAC)and reduced glutathione(GSH)are effective in reversing flupirtine-induced hepatotoxicity and whether they have other beneficial effects when combined with flupirtine.METHODS The analgesic effects of NAC and flupirtine were first evaluated in carrageenaninduced inflammatory pain and paclitaxel-induced neuropathic pain.The combination subthreshold⁃ing approach was then used to determine whether the combination of NAC and flupirtine produced synergistic analgesic effects.Hepatotoxicity markers and histopathological examination of the liver were used to assess the efficacy of NAC and GSH in reversing flupirtine-induced hepato⁃toxicity.Finally,the effect of GSH on the safe range of flupirtine was assessed in an acute tox⁃icity assay.RESULTS Flupirtine and NAC pro⁃duced dose-dependent antiallodynic effects evoked by carrageenan and paclitaxel in mice.In the above model,the combination of NAC and flupirtine produced an unexpected synergistic analgesic effect.There were no significant differ⁃ences observed in the hepatotoxicity markers and liver histopathology between the experimen⁃tal group and the control group under NAC and GSH treatment.Finally,GSH(200 mg·kg^(-1))expanded the therapeutic index of flupirtine by 1.77 times.CONCLUSION NAC and GSH are effective in preventing liver damage caused by long-term flupirtine use,which provides a solu⁃tion for the safe and effective treatment of chronic pain with flupirtine.In addition,the other benefi⁃cial effects of NAC and GSH when combined with flupirtine may provide the basis for the devel⁃opment of a new therapy with minimal sideeffects and good efficacy.

Evaluation of combined detection of nuclear factor erythroid 2-related factor 2 and glutathione peroxidase 4 in primary hepatic carcinoma and preliminary exploration of pathogenesis

This study aims to analyze the clinical significance and mechanism of nuclear factor erythroid 2-related factor 2(NRF2)and glutathione peroxidase 4(GPX4)in primary hepatic carcinoma(PHC).Methods:The expression of NRF2 and GPX4 in peripheral blood of patients with PHC was determined to analyze the diagnostic value of the two combined for PHC.The prognostic significance of NRF2 and GPX4 was evaluated by 3-year followup.Human liver epithelial cells THLE-2 and human hepatocellular carcinoma cells HepG2 were purchased,and the expression of NRF2 and GPX4 in the cells was determined.NRF2 and GPX4 aberrant expression vectors were constructed and transfected into HepG2,and changes in cell proliferation and invasion capabilities were observed.Results:The expression of NRF2 and GPX4 in patients with PHC was higher than that in patients with LC or VH(p<0.05),and the two indicators combined was excellent in diagnosing PHC.Moreover,patients with high expression of NRF2 and GPX4 had a higher risk of death(p<0.05).In in vitro experiments,both NRF2 and GPX4 expression was elevated in HepG2(p<0.05).HepG2 activity was enhanced by increasing the expression of the two,vice versa(p<0.05).Conclusion:NRF2 and GPX4 combined is excellent in diagnosing PHC,and promotes the malignant development of PHC.

Evaluation of Glutathione Peroxidase Enzymatic Activity in Seminal Plasma of Patients Treated at the Institute Pasteur in Cote d’Ivoire

Glutathione peroxidase (GPx) is an antioxidant that plays an important role in the maintenance of male fertility. The aim of this study was to compare the profile of enzymatic activity of glutathione peroxidase in the seminal plasma of normozoosperm and those of pathological sperm. Thus, the activity of glutathione peroxidase was determined in the seminal plasma of 20 normozoosperms, 9 azoosperms and 31 oligoasthenoteratozoosperms. It was 37.58 ± 3.14 U/L in normozoosperms, 39.39 ± 2.27 U/L in oligoasthenoteratozoosperms, and 29.77 ± 2.62 U/L in azoosperms. The mean GPx enzyme activity of normozoosperms did not differ significantly from that of oligoasthenoteratozoosperms and azoosperms. In contrast, comparison of enzyme activity between abnormal sperms gave a significant difference. This study showed that glutathione peroxidase enzymatic activity is not related to sperm quality.

Glutathione peroxidase mimicry of diphenyl diselenide: Plausible contribution of proteins’ thiols

Organoseleniums are a class of compounds attracting attention across the globe owing to their Glutathione peroxidase(GPx)mimicry,which confers on them a strong antioxidant activity.Diphenyl diselenide(DPDS)is an Organoselenium whose GPx mimetic property has been suggested to rely on the oxidation of non-protein or protein thiols critical to the activities of some sulfhydryl enzymes.This study,therefore investigated the GPx mimic/antioxidant property of DPDS as well as the role of thiols of two key sulfhydryl enzymes,cerebral Na^(+)/K^(+)-ATPase(sodium pump)and hepatic delta-aminolevulinic acid dehydratase(δ-ALAD)in the GPx mimicry of DPDS.Albino Wistar rats were euthanized,and the liver and brain were removed and used to assay for the effect of DPDS on lipid peroxidation induced by two prooxidants[Fe2^(+)(10μM)and H2O2,(1 mM)]as well as the activities of the sulfhydryl enzymes.The results revealed that DPDS profoundly(P<0.05)counteracted Fe2^(+)and H2O2-induced lipid peroxidation in the rats’hepatic and cerebral tissues.Furthermore,the results of assay systems for lipid peroxidation and sodium pump revealed that DPDS inhibited Na^(+)/K^(+)-ATPase and lipid peroxidation in the brain tissue homogenates in the same reaction system.A similar result was obtained in the assay system for lipid peroxidation and hepaticδ-ALAD as DPDS simultaneously inhibited the enzyme’s activity and lipid peroxidation.This suggests that the GPx mimetic property of DPDS may be linked to the enzymes’loss of activity,which further validates the suggestions that the enzymes’inhibition,as well as the antioxidant action of DPDS,rely on the oxidation of critical thiols of the enzymes.However,the GPx mimicry of DPDS should be investigated in the presence of thiol-blocking or oxidizing agents in biological systems in order to further ascertain the role of protein thiols.

Electronic Aspects of the Synergistic Antioxidant Interaction of Various Pairs “Phenolic Food Acid and Glutathione” in Their Reactions with the Stable Radical Cation ABTS+·

In the present work, for the first time, the main details of the electronic mechanism of the synergistic antioxidant interaction between different pairs: phenolic food acid and glutathione and the stable radical cation ABTS+· were revealed on the basis of a rigorous analysis of the DFT calculated data. It was shown that among all the studied food acids, only caffeic acid exhibits a clear-cut significant synergistic effect with glutathione. It established the electronic and structural factors underlying the mechanism of the synergistic interaction of the mixture caffeic acid and glutathione in its reaction with ABTS+·. The main causes of this considered synergistic effect are, firstly, the presence of the 3-OH and 4-OH hydroxyl groups in the structure of caffeic acid, secondly, the greater stability of its anion which contains the deprotonated 4-OH hydroxyl group. All other phenolic food acids under study do not possess the given structural particularity and therefore do not show such synergistic effects with glutathione.

双核Pt(Ⅳ)配合物与Guanosine-5′-Monophosphate和Glutathione反应的核磁共振光谱研究(英文)

合成了一个新型的双核Pt(Ⅳ)配合物｛[cis-Pt(NH3)2Cl(OH)2]2(4,4′-methylenedianiline)｝(NO3)2(化合物1)及相应的15N标记化合物｛[cis-Pt(15NH3)2Cl(OH)2]2(4,4′-methylenedianiline)｝(NO3)2(化合物15N-1)。利用1HNMR和ESMS进行了结构表征,化合物15N-1的2D[1H,15N]HSQCNMR发现,该化合物在水溶液中存在同分异构体。2D[1H,15N]HSQCNMR技术跟踪了化合物15N-1与Guanosine-5′-Monophosphate(5′-GMP)和Glutathione(GSH)的反应。结果显示,5′-GMP能在0.5h内将化合物1还原,而GSH在6h以后才能够部分的将化合物1还原。化合物1所表现出来的反应性能将有利于提高其治疗效果和降低毒副作用。

Response of Glutathione and Glutathione S-transferase in Rice Seedlings Exposed to Cadmium Stress

A hydroponic culture experiment was done to investigate the effect of Cd stress on glutathione content （GSH） and glutathione S-transferase （GST, EC 2.5.1,18） activity in rice seedlings. The rice growth was severely inhibited when Cd level in the solution was higher than 10 mg/L. In rice shoots, GSH content and GST activity increased with the increasing Cd level, while in roots, GST was obviously inhibited by Cd treatments, Compared with shoots, the rice roots had higher GSH content and GST activity, indicating the ability of Cd detoxification was much higher in roots than in shoots. There was a significant correlation between Cd level and GSH content or GST activity, suggesting that both parameters may be used as biomarkers of Cd stress in rice.

Induction of Glutathione and Glutathione S-transferase in Several Crops with the Treatment of Acetochlor

The response of glutathione(GSH) content and glutathione S-transferease(GST) activity to the acetochlor in roots and shoots of the maize 'Dongnong248',the sorghum 'Aoza No.2' and millet'Yugu' was evaluated.The concentrations of pre-emergence acetochlor causing a 50% inhibition of plant shoot height were 25 μmol·L^(-1) for the tolerant 'Dongnong248' maize,5 μmol·L^(-1) for the sensitive 'Aoza No.2' sorghum and 0.5 μmol·L^(-1) for the very sensitive 'Yugu'millet.Pre-treatment with 10 μmol·L^(-1) of acetochlor induced the root GST activities and nonprotein thiol content of all three cultivars.The induction of root GST activities and nonprotein thiol content compared to controls are observed on the fourth day after acetochlor treatment,The extents of activity and content increase from the higher to the lower were:tolerant maize cultivar 'Dongnong248'>sorghum cultivar'Aoza No.2'>millet cultivar 'Yugu'.The activities and contents induced in shoots were similar to that in roots,but the degrees of increase were less.Under different concentration treatment,the thiol content and GST activities increased with the herbicide concentration rising,then reached their peaks and began to decrease in all tested crop seedlings.The extent of induced GST activities and thiol content correlated well with differential cultivar resistance to acetochlor,so their protective mechanism appears to be strongly dependent on the endogenous levels of GSH and activities of GST.

Novel functional association of rat testicular membrane-associated cytosolic glutathione S transferases and cyclooxygenase in vitro

Aim:To analyze the role of cytosolic glutathione S-transferases (cGSTs) and membrane-associated cytosolic GSTs (macGSTs) in prostaglandin biosynthesis and to evaluate the possible interaction between glutathione S-transferases (GSTs) and cyclooxygenase (COX) in vitro.Methods:SDS-PAGE analysis was undertaken for characterization of GSTs,thin layer chromatography (TLC) to monitor the effect of GSTs on prostaglandin biosynthesis from arachi- donic acid (AA) and spectrophotometric assays were done for measuring activity levels of COX and GSTs.Results: SDS-PAGE analysis indicates that macGSTs have molecular weights in the range of 25-28 kDa.In a coupled assay involving GSTs,arachidonic acid and cyclooxygenase-1,rat testicular macGSTs produced prostaglandin E2 and F2~, while the cGSTs caused the generation of prostaglandin D2,E2 and F_(2α).In vitro interaction studies on GSTs and COX at the protein level have shown dose-dependent inhibition of COX activity by macGSTs and vice versa.This effect, however,is not seen with cGSTs.The inhibitory effect of COX on macGST activity was relieved with increasing concentrations of reduced glutathione (GSH) but not with 1-chloro 2,4-dinitrobenzene (CDNB).The inhibition of COX by macGSTs,on the other hand,was potentiated by glutathione.Conclusion:We isolated and purified macGSTs and cGSTs from rat testis and analyzed their involvement in prostaglandin biosynthesis.These studies reveal a revers- ible functional interaction between macGSTs and COX in vitro,with possible interactions between them at the GSH binding site of macGSTs.

Novel insights into conjugation of antitumor-active unsymmetrical bisacridine C-2028 with glutathione:Characteristics of non-enzymatic and glutathione S-transferase-mediated reactions

Unsymmetrical bisacridines(UAs) are a novel potent class of antitumor-active therapeutics.A significant route of phase II drug metabolism is conjugation with glutathione(GSH),which can be non-enzymatic and/or catalyzed by GSH-dependent enzymes.The aim of this work was to investigate the GSHmediated metabolic pathway of a representative UA,C-2028.GSH-supplemented incubations of C-2028 with rat,but not with human,liver cytosol led to the formation of a single GSH-related metabolite.Interestingly,it was also revealed with rat liver microsomes.Its formation was NADPH-independent and was not inhibited by co-incubation with the cytochrome P450(CYP450) inhibitor 1-aminobenzotriazole.Therefore,the direct conjugation pathway occurred without the prior CYP450-catalyzed bioactivation of the substrate.In turn,incubations of C-2028 and GSH with human recombinant glutathione S-transferase(GST) P1-1 or with heat-/ethacrynic acid-inactivated liver cytosolic enzymes resulted in the presence or lack of GSH conjugated form,respectively.These findings proved the necessary participation of GST in the initial activation of the GSH thiol group to enable a nucleophilic attack on the substrate molecule.Another C-2028-GSH S-conjugate was also formed during non-enzymatic reaction.Both GSH S-conjugates were characterized by combined liquid chromatography/tandem mass spectrometry.Mechanisms for their formation were proposed.The ability of C-2028 to GST-mediated and/or direct GSH conjugation is suspected to be clinically important.This may affect the patient’s drug clearance due to GST activity,loss of GSH,or the interactions with GSH-conjugated drugs.Moreover,GST-mediated depletion of cellular GSH may increase tumor cell exposure to reactive products of UA metabolic transformations.

Theoretical Study on GSH Activation Mechanism of a New Type of Glutathione Transferase Gtt2

Glutathione transferases（GSTs） play an important role in the detoxification of xenobiotic/endobiotic toxic compounds. The α-, π-, and/l-classes of cytosolic GSTs have been studied extensively, while Gtt2 from Saccharo- myces cerevisiae, a novel atypical GST, is still poorly understood. In the present study, we investigated the gluta- thione（GSH） activation mechanism of Gtt2 using the density functional theory（DFT） with the hybrid functional B3LYP. The computational results show that a water molecule could assist a proton transfer between the GSH thiol and the N atom of His133. The energy barrier of proton transfer is 46.0 kJ/mol. The GSH activation mechanism and the characteristics of active site are different from those of classic cytosolic GSTs.

Plasma glutathione and oxidized glutathione level, glutathione/oxidized glutathione ratio, and albumin concentration in complicated and uncomplicated falciparum malaria

Objective: To compare the level of glutathione(GSH) and oxidized glutathione(GSSG),the ratio of GSH/GSSG and the concentration of albumin in plasma of patients with complicated and un-complicated falciparum malaria.Methods: This research was a cross sectional study using comparison analysis with the plasma GSH and GSSG, the ratio of plasma GSH/GSSG and the concentration of plasma albumin as variables. The complicated malaria patients were obtained from Dr. Saiful Anwar Hospital Malang, whereas uncomplicated malaria patients were obtained from the Regency of Pleihari South Kalimantan. Plasma GSH and GSSG levels were determined by the spectrophotometer at the wave length of 412 nm, whereas the concentration of albumin was determined by bromocresol green method in the p H of 4.1.Results: There were no significant differences between the level of plasma GSH and GSSG in complicated and uncomplicated malaria patients, as well as the ratio of plasma GSH/GSSG in the two groups(P = 0.373; P = 0.538; and P = 0.615, respectively, independent ttest). In contrast, the plasma albumin concentration in complicated malaria patients were significantly higher than uncomplicated malaria patients(P = 0.000, Mann Whitney U test).Conclusions: It can be concluded that the average of plasma GSH and GSSG level, also plasma GSH/GSSG ratio in complicated malaria are not different from uncomplicated malaria. Although plasma concentration of albumin in both groups is below the normal range,there is an increase in complicated malaria that might be as compensation of oxidative stress.

T-cell allorecognition of donor glutathione S-transferase T1 in plasma cell-rich rejection

AIM To investigate the role of glutathione S-transferase T1 donor-specific T lymphocytes in plasma cell-rich rejection of liver allografts.METHODS The study group included 22 liver transplant patients. Among them, 18 patients were mismatched for the glutathione S-transferase T1(GSTT1) alleles(don+/rec-), and 4 were matched(don+/rec+). Seven of the mismatched patients produced anti-GSTT1 antibodies and developed plasma cell-rich rejection(former de novo immune hepatitis). For the detection of specific Tlymphocytes, peripheral blood mononuclear cells were collected and stored in liquid nitrogen. The memory T cell response was studied by adding to the cell cultures to a mix of 39 custom-made, 15-mer overlapping peptides, which covered the entire GSTT1 amino acid sequence. The specific cellular response to peptides was analyzed by flow cytometry using the markers CD8, CD4, IL-4 and IFNγ.RESULTS Activation of CD8^+ T cells with different peptides was observed exclusively in the group of patients with plasma-cell rich rejection(3 out of 7), with production of IL-4 and/or IFNγ at a rate of 1%-4.92% depending on the peptides. The CD4^+ response was most common and not exclusive for patients with the disease, where 5 out of 7 showed percentages of activated cells from 1.24% to 31.34%. Additionally, two patients without the disease but with the mismatch had cells that became stimulated with some peptides(1.45%-5.18%). Highly unexpected was the finding of a double positive CD4^+CD8^(low) T cell population that showed the highest degree of activation with some of the peptides in 7 patients with the mismatch, in 4 patients with plasma cell-rich rejection and in 3 patients without the disease. Unfortunately, CD4^+CD8^(low) cells represent 1% of the total number of lymphocytes, and stimulation could not be analyzed in 9 patients due to the low number of gated cells. Cells from the 4 patients included as controls did not show activation with any of the peptides. CONCLUSION Patients with GSTT1 mismatch can develop a specific T-cell response, but the potential role of this response in the pathogenesis of plasma cell-rich rejection is unknown.

Relationship between Glutathione Metabolism and TMV Resistance in Tobacco

This study was conducted to explore the relationship between glntathione metabolism and tobacco mosaic vires （TMV） resistance, using a TMV resistant tobacco cultivar Yuyan 8 and another tobacco cuhivar NC89 which has the same genetic background with Yuyan 8 as the experimental materials. Bioinformatics anal- ysis on the transcriptome data obtained from high-throughput sequencing revealed that among pathways enriched with differentially expressed genes, glntathione met- abolic pathway was found in both cultivars infected with TMV, and glntathione metabolism was enhanced in Yuyan 8. We detected the changes in the transcription of glntathione S-transferase （GST） which is a key enzyme in glutathione metabolic pathway with quantitative PER, the enzyme activity of GST and the content of glutathione （GSH） with spectmphotometry, and the expression levels of the genes that are related to TMV resistance or involved in photosynthesis. The results showed that GST gene expression, enzyme activity and GSH content were all increased by TMV infection in both cultivars, and the increments in Yuyan 8 were more significant. The expression of GST and other genes relate to TMV resistance were verified under different sulfur conditions. The results showed that the expression of these genes changed in a similar pattern in the two eultivars after TMV inoculation in both ＋ S and - S treatments, and their expression in - S treatment was slightly lower than in ＋ S treatment. The expression of the four genes related to TMV resistance （GST, PRI-a, HSP90 and Catalase-3） was up-regulated, and was higher in Yuyan 8 than in NC89 after TMV inoculation. On the contrast, the expression of the two genes involved in photosynthesis （ PsbA and Photosystem II 10 kDa polypeptide） were down-regulated, and the decrease in Yuyan 8 was less than in NC89. The results indicate that in the cultivars resistant to TMV infection, enhanced glntathione metabolism is beneficial to cellar redox balance, and the stable expression of PsbA which encodes the PSII reaction center protein DI can re- duce the damae to nhotosvnthetic system.