谷胱甘肽磷脂氢过氧化物酶研究进展

谷胱甘肽磷脂氢过氧化物酶(PHGPx)是生物体内一种重要的抗氧化酶。它是一种硒依赖性蛋白,在谷胱甘肽(GSH)的参与下能特异性地还原磷脂氢过氧化物(PLOOH)和胆固醇氢过氧化物(ChOOH),从而保护生物膜免受过氧化损伤。它还是核酸等生物大分子的重要保护剂,并且在细胞凋亡调控中发挥作用。     

重组萝卜磷脂氢谷胱甘肽过氧化物酶在毕赤酵母优化表达初步纯化与鉴定

将萝卜磷脂氢谷胱甘肽过氧化物酶（RsPHGPx）基因插入到分泌表达载体pPIC9K中,转化巴斯德毕赤酵母GS115细胞,筛选具有G418抗性的单拷贝转化子。经过优化表达条件,RsPHGPx在1%甲醇、pH6.0、28℃条件下诱导60h后得到最大表达量,产率约为102 mg/L。通过硫酸铵分级沉淀、脱盐柱脱盐、凝胶过滤等纯化步骤,得到了90%以上纯度的RsPHGPx．活性分析显示纯化获得的RsPHGPx具有依赖于GSH的还原活性, 比活性为4.2μmol/min·mg,为获得大量RsPHGPx而用于应用开发研究奠定了基础。     

萝卜磷脂氢谷胱甘肽过氧化物酶基因结构及其调控序列分析

磷脂氢谷胱甘肽过氧化物酶 (PHGPx) 是谷胱甘肽过氧化物酶 (GPx) 家族的重要一员,是目前已知能直接保护生物膜免受过氧化损伤的唯一酶类 . 此前的研究表明,萝卜磷脂氢谷胱甘肽过氧化物酶基因 (RsPHGPx) 编码一个有生理功能的过氧化物酶 , 并且 RsPHGPx 基因的表达可能受发育和环境胁迫信号的复杂调控 . 要深入了解该基因的表达调控机制首先必须阐明 RsPHGPx 基因的结构及其上游调控序列 . DNA 印迹表明萝卜 RsPHGPx 基因以单拷贝的形式存在于基因组中 . 以基因组 DNA 为模板,通过常规 PCR 与染色体步行相结合的方法克隆到了一段 3.3 kb 长的 RsPHGPx 基因组序列 . 分析发现,该基因由 7 个外显子和 6 个内含子组成,所有内含子的剪切位点均符合真核生物 GT-AG 规则 . 另外还发现该基因的上游基因是生物素合成酶基因;位于 RsPHGPx 基因上游的调控序列只有不足 300 bp. 这些结构特征与拟南芥 AtGPX3 基因极其相似 . 顺式作用元件的数据库搜索发现 RsPHGPx 基因的上游调控序列含有多个响应激素 ( 如 E-Box 和 W-Box) 、胁迫 ( 如转录因子 MYB 和 MYC 的结合位点 ) 和光 ( 如 Box Ⅱ和Ⅰ -Box) 信号的元件 . RNA 印迹分析表明 RsPHGPx 基因的表达受到脱落酸 (ABA) 和连续光照 ( 在黄化苗中 ) 处理的负调控,受到冷胁迫 (4℃ ) 的正调控,这暗示了预测的顺式作用元件的调控作用 . 然而,除草剂 paraquat 对该基因表达的正调控作用,暗示了某些与氧化胁迫相关的未知元件的存在 . 这些结果进一步印证了 RsPHGPx 基因的表达受发育和环境胁迫信号复杂调控的推测 . 这是迄今为止首个关于植物 PHGPx 基因结构和上游调控序列的系统报道,为今后全面认识植物 PHGPx 基因的表达调控机制奠定了必要基础 .     

原核表达的萝卜PHGPx对黑色素瘤B16细胞紫外辐射损伤的恢复作用

将大肠杆菌中高效表达的萝卜PHGPx（RsPHGPx）和突变的RsPHGPx（mRsPHGPx）融合蛋白,经Glutathione Sepharose 4B柱亲和纯化和PreScission蛋白酶柱上酶切,获得了不含GST标签的RsPHGPx和mRsPHGPx重组蛋白,纯度在90%以上。酶活测定验证了活性中心Cys突变的mRsPHGPx的活性显著低于RsPHGPx。考察RsPHGPx和mRsPHGPx对紫外辐射损伤后的黑色素瘤B16细胞的恢复作用,发现活性较高的RsPHGPx能有效提高细胞存活率,降低细胞膜脂质过氧化水平,而活性极低的mRsPHGPx则几乎没有效果,提示由于RsPHGPx能快速清除多种脂类过氧化物,可能主要通过抑制紫外辐射造成的细胞膜脂质过氧化损伤起作用。     

外源DMSO和ASA对微囊藻毒素胁迫下烟草细胞ROS生成和抗氧化系统的影响

采用2μg/mL微囊藻毒素-RR(MC-RR)、2μg/mL MC-RR 0.5%二甲基亚砜(DMSO)和2μg/mL MC-RR 2 mmol/L抗坏血酸(ASA)分别处理烟草悬浮细胞,研究上述各处理对烟草悬浮细胞活性氧(ROS)产生和抗氧化系统的影响。结果表明,与对照相比,MC-RR单独处理后烟草悬浮细胞中ROS、膜脂过氧化产物丙二醛(MDA)和细胞内源ASA的含量及超氧化物歧化酶(SOD)和过氧化物酶(POD)的活性明显升高,还原型谷胱甘肽(GSH)的含量有一个先降后升的变化过程。在分别加入外源抗氧化剂DMSO或ASA后,细胞内ROS和MDA含量下降,ASA、GSH含量和SOD、POD酶活性基本可恢复到对照水平。以上结果说明,微囊藻毒素单独处理细胞可造成氧化胁迫,其所诱导的ROS的大量积累很有可能是其产生细胞毒害的关键因子,外源抗氧化剂ASA和DMSO可缓解MC-RR对细胞的毒害作用,对细胞起一定保护作用。     

山楂叶总黄酮对游离脂肪酸损伤的胰岛βTC3 细胞的保护作用

目的:观察山楂叶总黄酮对棕榈酸损伤的胰岛βTC3细胞是否具有保护作用,并筛查出山楂叶总黄酮所起作用的有效浓度。方法:以胰岛βTC3细胞为研究对象,使用棕榈酸制作脂毒性模型,采用MTT法观察山楂叶总黄酮是否具有保护作用,并进一步筛查出有效浓度及时间;同时采用末端脱氧核苷酸转移酶介导的dUTP原位切口末端标记(TUNEL)技术检测胰岛βTC3细胞的凋亡情况。结果:MTT结果显示10-100μg/ml的山楂叶总黄酮对棕榈酸损伤的胰岛βTC3细胞均有保护作用,其吸光度值明显比棕榈酸组高(P<0.05),并且50μg/ml的山楂叶总黄酮与棕榈酸共同处理胰岛βTC3细胞24小时具有最好的保护作用;TUNEL检测结果显示山楂叶总黄酮+棕榈酸组胰岛βTC3细胞的凋亡率比棕榈酸组低(P<0.01)。结论:山楂叶总黄酮对脂毒性损伤的胰岛βTC3细胞具有保护作用,并在10-100μg/ml浓度范围内成一定的剂量依赖效应。     

二氢青蒿素通过诱导铁死亡抑制肝癌细胞生长

二氢青蒿素（dihydroartemisinin,DHA）是青蒿素的一种衍生物,在多种肿瘤中表现出明显的抗肿瘤活性,但其具体机制不详。本文探讨了DHA对肝癌细胞的毒性作用机制。利用CCK-8试剂检测DHA对肝癌细胞株活力的影响,通过荧光探针染色及流式细胞术分析细胞内ROS及脂质过氧化物水平的变化;通过谷胱甘肽测定试剂盒检测细胞内还原型谷胱甘肽含量的变化,并通过免疫印迹分析DHA作用下细胞内铁死亡通路蛋白质中GPX4的变化。结果发现,DHA能显著抑制SMMC-7721及Huh-7细胞活力,其半数抑制浓度分别为23.74 μmol/L及26.92 μmol/L。 在35 μmol/L DHA 处理下,SMMC-7721及Huh-7细胞内ROS分别升高2.6倍和2.1倍,脂质过氧化物升高2.3倍和1.7倍。DHA可诱导细胞内GSH含量下降,并能下调铁死亡相关蛋白质GPX4蛋白水平。通过利用小分子抑制剂进行功能恢复实验发现,ROS抑制剂、铁螯合剂及铁死亡抑制剂都可不同程度恢复DHA引起的细胞活力下降。进一步检测发现,铁死亡抑制剂可抑制DHA诱导的脂质过氧化,并恢复GSH含量及GPX4蛋白水平。结果表明,在肝癌细胞中,DHA可通过诱导细胞发生铁死亡抑制肝癌细胞生长。     

二氢青蒿素通过诱导铁死亡抑制肝癌细胞生长

二氢青蒿素（dihydroartemisinin,DHA）是青蒿素的一种衍生物,在多种肿瘤中表现出明显的抗肿瘤活性,但其具体机制不详。本文探讨了DHA对肝癌细胞的毒性作用机制。利用CCK-8试剂检测DHA对肝癌细胞株活力的影响,通过荧光探针染色及流式细胞术分析细胞内ROS及脂质过氧化物水平的变化;通过谷胱甘肽测定试剂盒检测细胞内还原型谷胱甘肽含量的变化,并通过免疫印迹分析DHA作用下细胞内铁死亡通路蛋白质中GPX4的变化。结果发现,DHA能显著抑制SMMC-7721及Huh-7细胞活力,其半数抑制浓度分别为23.74 μmol/L及26.92 μmol/L。 在35 μmol/L DHA 处理下,SMMC-7721及Huh-7细胞内ROS分别升高2.6倍和2.1倍,脂质过氧化物升高2.3倍和1.7倍。DHA可诱导细胞内GSH含量下降,并能下调铁死亡相关蛋白质GPX4蛋白水平。通过利用小分子抑制剂进行功能恢复实验发现,ROS抑制剂、铁螯合剂及铁死亡抑制剂都可不同程度恢复DHA引起的细胞活力下降。进一步检测发现,铁死亡抑制剂可抑制DHA诱导的脂质过氧化,并恢复GSH含量及GPX4蛋白水平。结果表明,在肝癌细胞中,DHA可通过诱导细胞发生铁死亡抑制肝癌细胞生长。     

维生素C和B_1对铅致小鼠睾丸损伤保护作用研究

目的探讨维生素C、B1联合用药拮抗铅对睾丸的毒性作用。方法 1.30只雄性小鼠随机分为2组(15只/组):空白对照组和铅染毒组。染铅组雄性小鼠每日腹腔注射醋酸铅20 mg/kg,空白对照组给予等容量的生理盐水,一日一次,第42日结束以上实验。将小鼠取血,用试剂盒检测血浆中谷胱甘肽的含量、谷胱甘肽过氧化物酶(GPx)和谷胱甘肽还原酶(GR)的活性。将睾丸组织经过处理后,在光镜及透射电镜下观察睾丸组织结构变化;2.45只雄性小鼠随机分为3组(15只/组):空白对照组、铅染毒组和维生素联合干预组。染铅组雄性小鼠每日腹腔注射醋酸铅20 mg/kg,维生素干预组在染铅后,即时每组小鼠给予维生素C和维生素B1,一日一次,第42日结束。处死小鼠,取血,用试剂盒检测血浆中谷胱甘肽的含量,谷胱甘肽过氧化物酶(GPx)和谷胱甘肽还原酶(GR)的活性。在光镜以及透射电镜下观察睾丸组织结构变化。结果1.与空白对照组比较,醋酸铅染毒组小鼠血浆中还原型谷胱甘肽(GSH)的水平降低,GSH/GSSG的比例降低,GPX活性升高,GR活性降低;光镜下睾丸组织生精小管变薄,各级生精细胞、支持细胞减少,部分细胞核出现核固缩;3.电镜下染毒组小鼠睾丸组织超微结构变化明显,支持细胞溶酶体增多,线粒体肿胀空泡化;2.与醋酸铅染毒组比较,维生素干预组小鼠血浆中还原型谷胱甘肽(GSH)的水平升高,GSH/GSSG的比例升高,GPx活性降低,GR活性升高;光镜观察结果:与染铅组相比,维生素B1和C联合干预组中破坏的睾丸生精上皮有所恢复;电镜观察结果:与染铅组相比,维生素B1和C联合干预组睾丸组织超微结构基本恢复正常,生精细胞和支持细胞结构正常,细胞器丰富。结果 1.醋酸铅使体内GSH的水平降低,GPX活性升高,GR活性降低,从而诱导氧化应激的发生,导致染铅小鼠睾丸的损伤;2.维生素干预组小鼠血浆中还原型谷胱甘肽(GSH)的水平升高,GSH/GSSG的比例升高,GPx活性降低,GR活性升高,维生素干预组小鼠血浆中氧化型谷胱甘肽还原为还原型谷胱甘肽,从而发挥抗氧化作用,对小鼠睾丸起到保护作用。     

谷胱甘肽与富氢冻存液在鸡血细胞冻存中的保护作用

目的通过观察细胞冷冻复苏后的存活率及凋亡情况,探讨细胞冻存液中添加谷胱甘肽(GSH)和氢气(H_2)对细胞的保护作用。方法在冻存液中添加GSH和/或通入H_2后冻存鸡血细胞,1个月后复苏,采用台盼蓝拒染和MTT法分别检测细胞存活率与细胞活性,流式细胞仪检测细胞的凋亡率。结果 GSH、H_2或H_2+GSH处理使细胞活力明显提高,而早期凋亡率明显降低。结论谷胱甘肽与富氢冻存液对鸡血细胞冻存具有保护作用。     

Contrasting Antioxidant and Cytotoxic Effects of Peroxiredoxin I and II in PC12 and NIH3T3 Cells

We examined the impact of peroxiredoxin-I (Prx-I) and peroxiredoxin-II (Prx-II) stable transduction on oxidative stress in PC12 neurons and NIH3T3 fibroblasts and found variability depending on cell type and Prx subtype. In PC12 neurons, Prx-II suppressed reactive oxygen species (ROS) generation by 36% (p < 0.01) relative to vector-infected control cells. However, in NIH3T3 fibroblasts, Prx-II overexpression resulted in a 97% (p < 0.01) increase in ROS generation. Prx-I transduction elevated ROS generation in PC12 cells. The effect of Prx-I on PC12 cells was potentiated in the presence of menadione, and suppressed by an inhibitor of nitric oxide synthetase. Prx-II transduction resulted in 25–35% lower levels of glutathione (GSH) in both cell types, while Prx-I transduction increased GSH levels in neurons and decreased GSH and caspase-3 activity in fibroblasts. Prx-I and Prx-II also had differing effects on cell viability. These results suggest that Prx-I and Prx-II can either increase or decrease intracellular oxidative stress depending on cell type or experimental conditions, particularly conditions affecting nitric oxide levels.Equivalent contributions were made by each author     

Modulation of antioxidant enzymes,reactive oxygen species,and glutathione levels in manganese superoxide dismutase-overexpressing NIH/3T3 fibroblasts during the cell cycle

NIH/3T3 mouse embryo fibroblasts were transfected with the cDNA for manganese superoxide dismutase (MnSOD). Previous studies showed characteristic unique AE profiles in nonsynchronous populations of parental, control plasmid-transfected, and MnSOD-overexpressing NIH/3T3 cell lines. However, the present study showed that during S and M phases of the cell cycle, antioxidant enzyme (AE) levels were altered in MnSOD-overexpressing cell lines towards levels in S and M phases of parental and control plasmid-transfected cells. Because of the demonstration that MnSOD overexpression inhibits cell growth in both nonmalignant and malignant cells, the present study was designed to measure AEs, reactive oxygen species (ROS), and glutathione levels in various phases of the cell cycle in both parental NIH/3T3 cells and NIH/3T3 cells overexpressing MnSOD, to try to determine whether AEs, ROS, and glutathione levels could have a possible regulatory role in cell cycle progression. In all cell lines studied, ROS levels were lower in M than S phase of the cell cycle. Total glutathione and glutathione disulfide levels were greatly increased during the M phase of the cell cycle compared with quiescence and S phase in all cell lines studied. This suggests that oxidative stress exists in M phase of the cell cycle with total glutathione levels increased to decrease oxidative stress. Analysis of MnSOD-overexpressing cell clones showed a correlation of decreased cell growth with an increase in ROS in S phase of the cell cycle and a decrease in glutathione in mitosis. The data strongly suggest that specific levels of cell redox state are necessary for cells to successfully progress through the various phases of the cell cycle. J. Cell. Physiol. 177:148–160, 1998. © 1998 Wiley-Liss, Inc.     

Human selenium-containing single-chain variable fragment with glutathione peroxidase activity protects NIH3T3 fibroblast against oxidative damage

Ultraviolet B (UVB medium wave, 280–315 nm) induces cellular oxidative damage and apoptosis by producing reactive oxygen species (ROS). Glutathione peroxidase functions as an antioxidant by catalyzing the reduction of hydrogen peroxide, the more important member of reactive oxygen species. A human selenium-containing single-chain variable fragment (se-scFv-B3) with glutathione peroxidase activity of 1288 U/μmol was generated and investigated for its antioxidant effects in UVB-induced oxidative damage model. In particular, cell viability, lipid peroxidation extent, cell apoptosis, the change of mitochondrial membrane potential, caspase-3 activity and the levels of intracellular reactive oxygen species were assayed. Human se-scFv-B3 protects NIH3T3 cells against ultraviolet B-induced oxidative damage and subsequent apoptosis by prevention of lipid peroxidation, inhibition of the collapse of mitochondrial membrane potential as well as the suppression of the caspase-3 activity and the level of intracellular ROS. It seems that antioxidant effects of human se-scFv-B3 are mainly associated with its capability to scavenge reactive oxygen species, which is similar to that of the natural glutathione peroxidase.     

Glutathione regulates telomerase activity in 3T3 fibroblasts

Changes in telomerase activity have been associated either with cancer, when activity is increased, or with cell cycle arrest when it is decreased. We report that glutathione, a physiological antioxidant present at high intracellular concentrations, regulates telomerase activity in cells in culture. Telomerase activity increases in 3T3 fibroblasts before exponential cell growth. The peak of telomerase activity takes place 24 h after plating and coincides with the maximum levels of glutathione in the cells. When cells are treated with buthionine sulfoximine, which decreases glutathione levels in cells, telomerase activity decreases by 60%, and cell growth is delayed. Glutathione depletion inhibits expression of E2F4 and Id2, which regulate the cell cycle. When glutathione levels are restored after incubation with glutathione monoethylester, telomerase activity and the cell cycle-related proteins return to control values. To discover the effect of glutathione redox status on the telomerase multicomplex structure, we incubated protein extracts from fibroblasts with different glutathione redox buffers. Telomerase activity is maximal under reduced conditions i.e. when the reduced/oxidized glutathione ratio is high. Consequently glutathione concentration parallels telomerase activity. These results underscore the main role of glutathione in the control of telomerase activity and of the cell cycle.     

STK15的表达对NIH3T3细胞的影响

目的构建pcDNA3.1-STK15表达质粒,探讨STK15基因对小鼠成纤维细胞（NIH3T3）的影响。方法构建pcDNA3.1-STK15质粒,将其转染NIH3T3,应用RT-PCR、免疫细胞化学和Western印迹方法检测STK15的表达;MTT法检测细胞增殖能力;Transwell检测细胞侵袭能力。结果转染pcDNA3.1-STK15质粒的NIH3T3细胞在48 h有STK15的表达,而且该细胞的增殖速度和穿透Matrigel胶的细胞数均明显高于对照组（P〈0.05）。结论STK15基因具有增加细胞增殖和细胞侵袭力的功能,进而形成肿瘤。     

RAF and antioxidants prevent cell death induction after growth factor abrogation through regulation of Bcl-2 proteins

We have shown previously that mitochondrial ROS production is essential to turn growth factor (GF) removal into cell death. Activated RAF, AKT, Bcl-2 and antioxidants protected equally well against ROS accumulation and subsequent death. Here we investigated whether protection by survival signaling and antioxidants utilizes shared or distinct targets. Using serum deprivation from NIH 3T3 fibroblasts and IL-3 withdrawal from promyeloid 32D cells, we showed that pro-survival signaling by activated RAF but not AKT prevented the decline in Mcl-1 following GF abrogation. GF starvation increased levels of Bim in both model systems, which was prevented by RAF in 32D cells but not in NIH 3T3 fibroblasts. RAF and AKT suppressed activation and mitochondrial translocation of BAX. Also, antioxidant treatment efficiently prevented BAX activation and death of 32D cells but showed little effect on its mitochondrial translocation. No significant impact of antioxidant treatment on Bim or Mcl-1 expression was observed. ROS produced during GF abrogation also did not alter the activity of intracellular signaling pathways, which have been implicated previously in cell killing by pro-oxidants. Together these data suggest Bcl-2 family proteins as convergence point for RAF and ROS in life and death decisions.     

Inhibition of cell growth in NIH/3T3 fibroblasts by overexpression of manganese superoxide dismutase: Mechanistic studies

NIH/3T3 mouse fibroblasts were transfected with the cDNA for manganese superoxide dismutase (MnSOD), and two clones overexpressing MnSOD activity were subsequently characterized by comparison with parental and control plasmid-transfected cells. One clone with a 1.8-fold increase in MnSOD activity had a 1.5-fold increase in glutathione peroxidase (GPX) activity (increased GPX-adapted clone), while a second clone with a 3-fold increase in MnSOD activity had a 2-fold decrease in copper, zinc superoxide dismutase (CuZnSOD) activity (decreased CuZnSOD-adapted clone). Increased reactive oxygen species (ROS) levels compared with parental or control plasmid-transfected cells were observed in nonsynchronous cells in the increased GPX-adapted clone, but not in the decreased CuZnSOD-adapted clone. The two MnSOD-overexpressing clones showed different sensitivities to agents that generate oxidative stress. Flow cytometry analysis of the cell cycle showed altered cell cycle progression in both MnSOD-overexpressing clones. During logarithmic growth, both MnSOD-overexpressing clones showed increased mitochondrial membrane potential compared with parental and control plasmid-transfected cells. Both MnSOD-overexpressing clones showed a decrease in mitochondrial mass at the postconfluent phase of growth, suggesting that mitochondrial mass may be regulated by MnSOD and/or ROS levels. Our results indicate that adaptation of fibroblasts to overexpression of MnSOD can involve more than one mechanism, with the resultant cell phenotype dependent on the adaptation mechanism utilized by the cell. J. Cell. Physiol. 175:359–369, 1998. © 1998 Wiley-Liss, Inc.     

The molecular mechanism of protecting cells against oxidative stress by 2-selenium-bridged beta-cyclodextrin with glutathione peroxidase activity

Ultraviolet B (UVB) induces apoptosis and lipid peroxidation of NIH3T3 cells by producing reactive oxygen species (ROS). Glutathione peroxidase (GPX) is one of the most important antioxidant enzymes in organism and it can scavenge ROS. 2-selenium-bridged beta-cyclodextrin (2-SeCD) is a GPX mimic generated in our lab. Its GPX activity is 7.4 U/mumol, which is 7.5 times as much as that of ebselen. In this paper, we have established a cell damage system using UVB radiation. Using this system, we have determined antioxidant effect of 2-SeCD by comparison of malondialdehyde (MDA) and H(2)O(2) contents in NIH3T3 cells before and after UVB radiation. Experimental results indicate that 2-SeCD can inhibit lipid peroxidation and protect the cells from the damage generated by UVB radiation. To evaluate the molecular mechanism of this protection, we determined the effect of 2-SeCD on the expression of p53 and Bcl-2 in NIH3T3 cells. The results showed that 2-SeCD inhibits the increase of p53 expression level and the decrease of expression of Bcl-2 induced by UVB radiation. Thus, we have concluded that protection of NIH3T3 cells against oxidative stress by 2-SeCD was carried out by regulation of the expression of Bcl-2 and p53.     

Cryoprotective effects of D-allose on mammalian cells

D-allose, an aldo-hexose, is a rare sugar whose biological functions remain largely unclear. Recently, we demonstrated a novel inhibitory effect of D-allose on production of reactive oxygen species (ROS). Here, we focused on investigating cryoprotective effects of D-allose on cell viability. Mammalian cell lines including OVCAR-3 (human ovarian cancer), HeLa (human cervical cancer), HaCaT (human skin keratinocytes), HDF (human dermal fibroblasts) and NIH3T3 (murine fibroblasts) cells were frozen at -80 degrees C in culture media with various D-allose concentrations. Cells were allowed to recover for 24 h, 1 week or 1 month prior to survival assessment using the trypan blue dye exclusion test, when cell proliferation was evaluated by MTT assay. A beneficial protective role of D-allose on cell survival was found, similar to that of trehalose (disaccharide of glucose), a recognized cryoprotectant. The results suggest that D-allose as a sole additive may provide effective protection for mammalian cells during freezing. Practical studies now need to be performed with D-allose, for example to determine optimal freezing protocols and explore potential for preservation of tissues or organs at non-freezing temperatures.     

The Depletion of Nuclear Glutathione Impairs Cell Proliferation in 3t3 Fibroblasts

Background

Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate.

Principal Findings

We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM) and buthionine sulfoximine (BSO), and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.

Conclusions

Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.