Measurement of mouse liver glutathione S-transferase activity by the integrated method

Objective: The integrated method was investigated to measure Vm/Km of mouse liver glutathione S-transfer-ase (GST) activity on GSH and 7-Cl-4-nitrobenzofurazozan. Methods: Presetting concentration of one substrate twenty-fold above the other's and taking maximum product absorbance Am as parameter while Km as constant, Vm/Km was obtained by nonlinear fitting of GST reaction curve to the integrated Michaelis-Menten equation In [Am/(Am -Ai)] + Ai/ ( ξ× Km ) = ( Vm/Km )×ti (1). Results: Vm/Km for GST showed slight dependence on initial substrate concentration and data range, but it was resistant to background absorbance, error in reaction origin and small deviation in presetting Km. Vm/Km was proportional to the amount of GST with upper limit higher than that by initial rate. There was close correlation between Vm/Km and initial rate of the same GST. Consistent results were obtained by this integrated method and classical initial rate method for the measurement of mouse liver GST. Conclusion: With the concentration of one substrate twenty-fold above the other's, this integrated method was reliable to measure the activity of enzyme on two substrates , and substrate concentration of the lower one close to its apparent Km was able to be used.

Antibacterial Activity of Exogenous Glutathione and Its Synergism on Antibiotics in Methicillin-Associated Multidrug Resistant Clinical Isolates of Staphylococcus aureus

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most problematic human pathogens. Antibiotic treatment of MRSA often associated with resistance to multiple classes of antibiotics is extremely challenging and urgently demands action to treat MRSA. Glutathione (GSH) is a biogenic thiol-compound that maintains an optimal intracellular redox-potential required for various normal cellular processes. Antibacterial activity of exogenous GSH has been reported in some bacterial pathogens but is largely unknown in MRSA. Aim: This study aimed to understand antibacterial activity of GSH, its role in antibiotic susceptibility, and a potential antibacterial mechanism in clinical isolates of S. aureus. Materials and Methods: Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), checkerboard, time-killing, and bacterial killing assays were performed for 14 clinical isolates of S. aureus including 10 MRSA and two type strains (ATCC 700699 and 35556). Results: MIC and MBC levels for the clinical and type strains were 15 - 20 mM and 25 - 40 mM of GSH, respectively. Subinhibitory concentrations of GSH synergistically enhanced susceptibility of all tested-antibiotics, resulting in sensitizing all-tested S. aureus. Bacterial-killing produced by GSH-mediated acidity was significantly higher than that by hydrochloric acid-mediated acidity. Conclusion: Overall results concluded that GSH exhibited antibacterial activity on S. aureus regardless of antibiotic susceptibility and synergistically enhanced antibiotic susceptibility. Additionally, GSH-mediated acidity was one of the antibacterial mechanisms. These findings suggest that GSH may be a potential antimicrobial agent or adjuvant for the conventional anti-MRSA regimens.

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.

Cloning, characterization and expression analysis of a microsomal glutathione S-transferase gene from the seagrass Zostera marina

The response of glutathione S-transferase(GST)in Zostera marina to temperature variation was analyzed at molecular level by cloning the microsomal GST gene and texting the microsomal GST expression regularity under different temperature.Specific speaking,express ZmGST in Escherichia coli,then purify the recombinant protein and make the thermal stability analysis.Therefore,the experiments were carried out to provide a theoretical basis for the further elaboration to the population degradation mechanisms of Z.marina.In conclusion,the thermostability and the response of ZmGST gene to temperature changes can determine its temperature tolerance range,and affect its resilience in turn.

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.

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.

Vanadium induces liver toxicity through reductive activation by glutathione and mitochondrial dysfunction

Pentavalent vanadium (V5+) (metavanadate salt) tox- icity is a challenging problem to the health professionals and has been recognized as an industrial hazard that adversely affects human and animal health, but its cytotoxic mechanisms have not yet been completely understood. In this study, we investigated the cytotoxic mechanisms of V5+ in freshly isolated rat hepatocytes. V5+ cytotoxicity was associated with reactive oxygen species (ROS) formation, collapse of mitochondrial membrane potential, lysosomal membrane rupture and cytochrome c release into the hepatocyte cytosol. All of the above mentioned V5+ -induced cytotoxicity markers were significantly (p 5+ is activated by GSH. Our findings also showed that the lysosomotropic agents prevented V5+ induced mitochondrial membrane potential collapse. On the other hand, mitochondrial MPT pore sealing agents inhibited lysosomal membrane damage caused by V5+. It can therefore be suggested that there is probably a toxic interaction (cross-talk) between mitochondrial and lysosomal oxidative stress generating systems, which potentiates ROS formation and further damages both sub-organelles in V5+-induced induced hepatotoxicity. In conclusion, V5+-induced cytotoxicity can be attributed to oxidative stress started from glutathione mediated metal reductive activation and continued by mitochondrial/lysosomal toxic interaction.

Polymorphism analysis of Glutathione S-transferase A1 in patients with hematological diseases and its effect on GST enzyme activity

Glutathione S-transferases(GSTs)are important drug-metabolizing enzymes that catalyze the binding of glutathione(GSH)to electrophilic substances.GST has genetic polymorphism,and the enzyme activity of GST affects the metabolism of certain drugs in vivo.In the present day,we investigated the GST enzyme activity and GSTA1 gene polymorphism in 170 patients with hematological diseases and explored their relationship.The GSTA1 gene polymorphism of the patient was analyzed by PCR-restriction fragment length polymorphism(PCR-RFLP)technique,and the base sequences of the four mutation sites(-631,-567,-69,and-52)in the promoter region were determined by DNA-Sequencer.The patient’s GST enzyme activity was calculated by measuring the rate at which it catalyzed the reaction between 1-chloro-2,4-dinitrobenzene(CDNB)and GSH.The average GST enzyme activities of males and females were 5.20±0.13 and 5.17±0.12 nmol/min/mL,respectively,and the difference was not significant(P=0.91).The frequencies of genotypes GSTA1*A*A(wild genotype),GSTA1*A*B(heterozygous genotype),and GSTA1*B*B(homozygous mutant genotype)were 75.3%,22.9%,and 1.8%,respectively.Alleles GSTA1*A and*B were distributed at 86.8%and 13.2%,respectively.The genotype frequency distribution between males and females was no significant difference by Pearson’s chi-square test(P=0.743).The average GST activity of the heterozygous mutant genotype(4.83±0.76 nmol/min/mL)was lower than the wild genotype(5.34±1.26 nmol/min/mL,P=0.018),and higher than that of the homozygous mutant genotype(3.32±0.07 nmol/min/mL,P=0.022).These findings might help us improve the individualized treatment of patients with hematological diseases in the future and promote the development of precision medicine for blood diseases.

Antioxidation Activities of Low-Molecular-Weight Gelatin Hydrolysate Isolated from the Sea Cucumber Stichopus japonicus

Gelatin extracted from the body wall of the sea cucumber （Stichopus japonicus） was hydrolyzed with flavourzyme. Low-molecular-weight gelatin hydrolysate （LMW-GH） of 700-- 1700 Da was produced using an ultrafiltration membrane bioreaetor system. Chemiluminescence analysis revealed that LMW-GH scavenges high free radicals in a concentration-dependent manner; IC50 value for superoxide and hydroxyl radicals was 442 and 285 μgmL-1, respectively. LMW-GH exhibited excellent inhibitory characteristics against melanin synthesis and tyrosinase activity in B16 cells. Furthermore, LMW-GH notably increased in- traeellular glutathione （GSH）, which in turn suppressed melanogenesis. LMW-GH performs antioxidation activity, holding the potential of being used as a valuable ingredient in function foods, cosmetics and pharmaceuticals or nutriceuticals.

Effects of PAHs on Biotransformation Enzymatic Activities in Fish

Biotransformation and detoxification responses to the exposure to five polycyclic aromatic hydrocarbons were investigated in crucian（Carassius auratus）. Juvenile crucian were treated with a single intraperitoneal injection of each compound at dosages of 0.1, 1.0, 2.0, 5.0 and 8.0（or 10.0） mg/kg and sacrificed 15 d later to determine 7-ethoxyresorufin-O-deethylase（EROD） and glutathione S-transferases（GST） activities in gill S9 fractions. EROD activity is significantly increased by benzo（b）fluoranthene and indeno（1,2,3-cd）pyrene at all the doses. High dosages of PAHs induced GST activity and the inducing ability of them increased in the following order： fluorene fluorantheneindeno（1,2,3-cd）pyrenebenzo（g,h,i）perylenebenzo（b）fluoranthene. In all the cases, dose dependence appeared to exist. The gill EROD and GST in Carassius auratus are useful biomarkers to estimate sub-acute toxicity of both polycyclic aromatic hydrocarbons（PAHs） and PAHs-like compounds.

Ultrathin FeS nanosheets with high chemodynamic activity for sensitive colorimetric detection of H_(2)O_(2) and glutathione

Iron chalcogenides have attracted great interest as potential substitutes of nature enzymes in the colorimetric biological sensing due to their unique chemodynamic characteristics.Herein,we report the preparation of ultrathin Fe S nanosheets(NSs)by a simple one-pot hydrothermal method and the prepared Fe S NSs exhibit strong Fenton-reaction activity to catalyze hydrogen peroxide(H_(2)O_(2))for generation of hydroxyl radical(^(·)OH).Based on the chromogenic reaction of resultant^(·)OH with 3,3,5,5-tetramethylbenzidine(TMB),we develop colorimetric biosensors for highly sensitive detection of H_(2)O_(2)and glutathione(GSH).The fabricated biosensors show wide linear ranges for the detection of H_(2)O_(2)(5–150μmol/L)and GSH(5–50μmol/L).Their detection limits for H_(2)O_(2)and GSH reach as low as0.19μmol/L and 0.14μmol/L,respectively.The experimental results of sensing intracellular H_(2)O_(2)and GSH demonstrate that this colorimetric method can realize the accurate detection of H_(2)O_(2)and GSH in normal cells(L02 and 3T3)and cancer cells(MCF-7 and He La).Our results have demonstrated that the synthesized Fe S NSs is a promising material to construct colorimetric biosensors for the sensitive detection of H_(2)O_(2)and GSH,holding great promising for medical diagnosis in cancer therapy.

Palladium Nanoparticles/Graphdiyne Oxide Nanocomposite with Excellent Peroxidase-like Activity and Its Application for Glutathione Detection

In this study, palladium nanoparticles loaded graphdiyne oxide (Pd/GDYO) nanocomposite were fabricated by in-situ reduction of palladium chloride in the dispersion of GDYO, and characte-rized by Raman spectra, transmission electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. The synthesized Pd/GDYO was first found to have catalytic activities similar to those of the peroxidase enzyme, which can catalyze the oxidation of peroxidase substrate 3,3',5,5'-tetramethylbenzidine(TMB) in the presence of hydrogen peroxide(H2O2). Steady-state kinetic studies showed that the catalytic reaction of Pd/GDYO follows a ping-pong mechanism, and Pd/GDYO has a stronger activity to TMB with a Michaelis constant(Km) value of 5.32×10-4 mmol/L. Based on the shielding effect of glutathione(GSH) on the Pd/GDYO-H2O2-TMB reaction system, a colorimetric detection method for GSH was deve-loped with a wide linear range from 0.1 μmol/L to 40 μmol/L and a limit of detection of 0.1 μmol/L. The method was successfully applied for fast and accurate detection of GSH in injection powder drugs. It was expected that this peroxidase-like Pd/GDYO nano- composite would have wide applications in the fields of biomedicine and environmental chemistry.

Effects of Different Cadmium Levels on Active Oxygen Metabolism and H_2O_2-Scavenging System in Brassica campestris L.ssp.chinensis

The effects of different Cd (Cadmium) levels on generation of active oxygen speceies(AOS) and H2O2-scavenging system in the leaves of Brassica campestris L. ssp. chinensiswere studied. The results showed that generation rate, and H2O2 content were enhancedand malondialdehyde (MDA) content increased with the increase of Cd concentrations inthe growth medium. The activities of ascorbate peroxidase (APX), dehydroascorbatereductase (DR) and glutathione reductase (GR) were promoted by the addition of Cd.Exposed to Cd also increased the contents of ascorbate (AsA) and glutathione (GSH) in theleaves.

外源谷胱甘肽对青花菜硫代葡萄糖苷合成的影响

为探究谷胱甘肽对硫代葡萄糖苷(GSLs)生物合成的影响,以现蕾期青花菜品种耐寒优秀为试验材料,研究不同浓度的还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)及丁硫堇(BSO)对青花菜花球中硫代葡萄糖苷及其相关底物含量、酶活性和基因表达的影响。结果表明,与CK(蒸馏水)相比,5 mg·L^(-1) GSH在处理48 h显著提高了青花菜花球中总硫苷、脂肪族硫苷含量,在24~48 h显著提高半胱氨酸(Cys)含量,在6~24 h显著提高了谷胱甘肽含量,在3~12 h显著提高了硫苷合成相关基因的表达量;45 mg·L^(-1) GSH处理在48 h显著降低了青花菜花球中总硫苷、脂肪族硫苷含量,在3~48 h则显著提高了半胱氨酸含量,在6~48 h则显著提高了谷胱甘肽含量,在3~12 h显著抑制了硫苷合成相关基因的表达。与CK相比,5 mg·L^(-1) GSSG处理下青花菜花球中总硫苷、脂肪族硫苷含量显著升高,而25、45及65 mg·L^(-1) GSSG处理则对总硫苷、脂肪族硫苷含量没有产生显著影响。综上所述,外源谷胱甘肽对硫苷含量的影响具有浓度效应,5 mg·L^(-1) GSH促进硫苷合成,45 mg·L^(-1) GSH抑制硫苷合成。

响应面法优化沙枣叶多酚提取工艺及体内外抗氧化活性研究

采用单因素实验和响应面法优化了新疆沙枣叶多酚的超声辅助提取工艺,并通过测定沙枣叶多酚对DPPH自由基的清除能力及对小鼠血清中丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、总抗氧化能力(T-AOC)的影响评价其抗氧化活性。结果表明,沙枣叶多酚的最佳提取工艺为:料液比1∶40(g∶mL)、乙醇体积分数58%、提取时间21 min、提取功率250 W,在此条件下,多酚含量为36.45 mg·g^(-1);当浓度为0.20 mg·mL^(-1)时,沙枣叶多酚和VC对DPPH自由基的清除率分别为90.3%、96.5%;与空白对照组比较,模型对照组小鼠血清中MDA水平极显著升高(P<0.01),SOD、GSH-Px、T-AOC的水平均极显著降低(P<0.01),且模型对照组的肾脏系数和肝脏系数与空白对照组差异极显著(P<0.01);与模型对照组比较,沙枣叶多酚高剂量组血清中MDA水平极显著降低(P<0.01),SOD、GSH-Px、T-AOC的水平均极显著升高(P<0.01),且肾脏系数和肝脏系数与模型对照组无显著差异(P>0.05)。

周氏啮小蜂CcGSTS1基因的克隆·蛋白表达纯化及酶学特征分析

鉴定一个编码周氏啮小蜂谷胱甘肽S-转移酶序列的全长基因CcGSTS1,系统发育分析发现,该基因与丽蝇蛹集金小蜂NvGSTS6基因具有高同源性。体外表达、纯化的重组CcGSTS1可催化CDNB与GSH结合,最适反应pH为7.0。该试验为进一步研究周氏啮小蜂CcGSTS1基因功能奠定基础。

Pharmacological Investigation on the Qi-Invigorating Action of Schisandrin B: Effects on Mitochondrial ATP Generation in Multiple Tissues and Innate/Adaptive Immunity in Mice

Schisandrae Fructus, containing schisandrin B (Sch B) as its main active component, is recognized in traditional Chinese medicine (TCM) for its Qi-invigorating properties in the five visceral organs. Our laboratory has shown that the Qi-invigorating action of Chinese tonifying herbs is linked to increased mitochondrial ATP generation and an enhancement in mitochondrial glutathione redox status. To explore whether Sch B can exert Qi-invigorating actions across various tissues, we investigated the effects of Sch B treatment on mitochondrial ATP generation and glutathione redox status in multiple mouse tissues ex vivo. In line with TCM theory, which posits that Zheng Qi generation relies on the Qi function of the visceral organs, we also examined Sch B’s impact on natural killer cell activity and antigen-induced splenocyte proliferation, both serving as indirect measures of Zheng Qi. Our findings revealed that Sch B treatment consistently enhanced mitochondrial ATP generation and improved mitochondrial glutathione redox status in mouse tissues. This boost in mitochondrial function was associated with stimulated innate and adaptive immune responses, marked by increased natural killer cell activity and antigen-induced T/B cell proliferation, potentially through the increased generation of Zheng Qi.

乙胺双碲醚的合成及GPx酶学性质研究

作为人体中一种非常重要的抗氧化酶,谷胱甘肽过氧化物酶(GPx)在清除人体生理反应产生的多余过氧化物中起到了非常重要的作用。由于人工智能GPx酶的催化活性较低,设计并合成出了同时含有GPx活性中心碲和两个伯氨基的乙胺双碲醚分子,当pH=7时活性为1.6±0.02μmol·min^(-1)·μmol^(-1),相较于经典小分子GPx人工硒酶提高了3个数量级。

灯盏花乙素抑制人前列腺癌细胞系PC-3的增殖和迁移

目的探讨灯盏花乙素(STR)对人前列腺癌细胞系(PC-3)增殖、迁移的影响及其作用机制。方法PC-3细胞分为STR低、中和高剂量组、colivelin(STAT3激活剂)组、STR高剂量+colivelin组、对照组。CCK-8法、集落形成实验检测细胞增殖;划痕实验检测细胞迁移;透射电镜观察细胞线粒体超微结构;比色法检测细胞内游离Fe 2+、丙二醛(MDA)含量及活性氧(ROS)水平;RT-qPCR检测溶质载体家族7成员11(SLC7A11)、增殖细胞核抗原(PCNA)、基质金属蛋白酶(MMP)-9 mRNA表达;Western blot检测p-STAT3、GPX4蛋白。结果与对照组对比,STR低、中和高剂量组细胞线粒体结构破坏明显,A 450值、集落形成率、划痕愈合率、PCNA、SLC7A11、MMP-9 mRNA表达及p-STAT3、GPX4蛋白降低(P<0.05),Fe 2+、MDA含量及ROS水平升高,且呈剂量依赖性(P<0.05);与对照组相比,colivelin组细胞中线粒体结构破坏有所改善,A 450值、集落形成率、划痕愈合率、PCNA、SLC7A11、MMP-9 mRNA表达及p-STAT3、GPX4蛋白升高,Fe 2+、MDA含量及ROS水平降低(P<0.05);与STR高剂量组相比,STR高剂量+colivelin组细胞中线粒体结构破坏有所减轻,A 450值、集落形成率、划痕愈合率、PCNA、SLC7A11、MMP-9 mRNA表达及p-STAT3、GPX4蛋白升高,Fe 2+、MDA含量及ROS水平降低(P<0.05)。结论STR降低和减弱前列腺癌细胞增殖和迁移能力,其机制可能与抑制STAT3/GPX4通路有关。

外源水杨酸对黄瓜叶片几种酶活性和抗氧化物质含量的影响

采用不同浓度的水杨酸 (SA)对营养液培养的黄瓜 (CucumissativusL .)进行处理 ,结果表明 ,不同浓度的SA均明显提高了黄瓜叶片中过氧化物酶、多酚氧化酶、苯丙氨酸解氨酶的活性 ,其中以SA 2 0 0 μmol·L-1 处理的效果最为显著。SA 1 0 0 μmol·L-1 的处理显著抑制了IAA氧化酶和抗坏血酸氧化酶的活性 ,而SA 2 0 0和 4 0 0 μmol·L-1 的处理则有促进作用。SA 1 0 0 μmol·L-1 的处理显著增加了黄瓜叶片中还原型抗坏血酸、还原型谷胱甘肽和氧化型谷胱甘肽的含量。