PTEN deletion leads to deregulation of antioxidants and increased oxidative damage in mouse embryonic fibroblasts

Despite the significance of oxidative damage in carcinogenesis, the molecular mechanisms that lead to increased susceptibility to oxidative stress are not well understood. We now report a link between loss of protection against oxidative damage and loss of function of PTEN, a highly mutated tumor suppressor gene in a variety of human tumors. Using two-dimensional gel electrophoresis, combined with Western and Northern blot analyses, we found that PTEN deficiency in mouse embryonic fibroblasts (MEFs) displays deregulated expression of several antioxidant enzymes, including peroxiredoxins 1, 2, 5, and 6 and Cu, Zn superoxide dismutase. In these Pten-deleted MEFs, the basal levels of reactive oxygen species (ROS) were increased, and both the basal level and the ROS-induced oxidative damage of DNA were increased, as evidenced by increased levels of hydrogen peroxide (H2O2), superoxide anion, 8-hydroxy-2'-deoxyguanosine, and DNA double-strand breaks. We further show that Pten deletion is correlated with resistance to H2O2-induced expression of several antioxidants. These findings suggest an essential role for PTEN in maintaining the normal redox state of mouse embryonic fibroblasts against oxidative damage. They also provide a molecular link between PTEN, whose inactivation is known to be involved in a variety of human tumors, and antioxidants, whose perturbation leads to oxidative damage of cells.     

Cooperation of phosphatidylcholine-specific phospholipase C and basic fibroblast growth factor in the neural differentiation of mesenchymal stem cells in vitro

Previously, we found that suppressing phosphatidylcholine-specific phospholipase C could induce neuronal differentiation of rat mesenchymal stem cells in the absence of serum and fibroblast growth factor. It is well known that basic fibroblast growth factor plays an important role in mesenchymal stem cell neuronal differentiation. In this study, our purpose was to understand the cooperation of phosphatidylcholine-specific phospholipase C and basic fibroblast growth factor in controlling mesenchymal stem cell neuronal differentiation. Our results showed that suppressing phosphatidylcholine-specific phospholipase C in the presence of basic fibroblast growth factor could induce cell neuronal differentiation and the viability of the differentiated cells was obviously increased. Furthermore, we found that the resting membrane potential of the differentiated cells gradually decreased, but the mitochondrial membrane potential rose with increasing treatment time and these characteristics were similar to cultured neurons from mouse embryo forebrains. To determine the possible mechanism by which this combination controls cell neuronal differentiation, we measured changes in the mitochondrial membrane potential and in the levels of reactive oxygen species. The results showed that both the mitochondrial membrane potential and reactive oxygen species levels decreased when basic fibroblast growth factor was added. The data suggested that lower phosphatidylcholine-specific phospholipase C activity was required for mesenchymal stem cell neuronal differentiation and basic fibroblast growth factor was necessary for maintaining the neuronal differentiation state. Moreover, basic fibroblast growth factor could contribute to rescuing the differentiated cells from death through decreasing overly high mitochondrial membrane potentials and reactive oxygen species levels.     

Derivation and maintenance of human embryonic stem cells from poor-quality in vitro fertilization embryos

Human embryonic stem (hES) cells are self-renewing, pluripotent cells that are valuable research tools and hold promise for use in regenerative medicine. Most hES cell lines are derived from cryopreserved human embryos that were created during in vitro fertilization (IVF) and are in excess of clinical need. Embryos that are discarded during the IVF procedure because of poor morphology and a low likelihood for generating viable pregnancies or surviving the cryopreservation process are also a viable source of hES cells. In this protocol, we describe how to derive novel hES cells from discarded poor-quality embryos and how to maintain the hES cell lines.     

In situ simultaneous organics and nitrogen removal from recycled landfill leachate using an anaerobic-aerobic process

An anaerobic-aerobic process including a fresh refuse landfill reactor as denitrifying reactor, a well-decomposed refuse reactor as methanogenesis reactor and an aerobic activated sludge reactor as nitrifying reactor was operated by leachate recirculation to remove organic and nitrogen simultaneously. The results indicated that denitrification and methanogenesis were carried out successfully in the fresh refuse and well-decomposed landfill reactors, respectively, while the nitrification of NH(4)(+)-N was performed in the aerobic reactor. The maximum organic removal rate was 1.78 kg COD/m(3)d in the well-decomposed refuse landfill reactor while the NH(4)(+)-N removal rate was 0.18 kg NH(4)(+)-N/m(3)d in the aerobic reactor. The biogas from fresh refuse reactors and well-decomposed refuse landfill reactors were consisted of mainly carbon dioxide and methane, respectively. The volume fraction of N(2) increased with the increase of NO(3)(-)-N concentration and decreased with the drop of NO(3)(-)-N concentration. The denitrifying bacteria mustered mainly in middle layer and the denitrifying bacteria population had a good correlation with NO(3)(-)-N concentration.     

Quantitative determination of mitiglinide in human plasma by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A selective, rapid and sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed for the quantitative determination of mitiglinide in human plasma. With nateglinide as internal standard, sample pretreatment involved a one-step extraction with diethyl ether of 0.2 mL plasma. The separation was performed on an ACQUITY UPLCtrade mark BEH C(18) column (50 mm x 2.1 mm, i.d., 1.7 microm) with the mobile phase consisting of methanol and 10 mmol/L ammonium acetate (65:35, v/v) at a flow rate of 0.25 mL/min. The detection was carried out by means of electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring (MRM). Linear calibration curves were obtained in the concentration range of 1.080-5400 ng/mL, with a lower limit of quantification of 1.080 ng/mL. The intra- and inter-day precision (RSD) values were below 15% and accuracy (RE) was from -3.5% to 7.3% at all QC levels. The method was fully validated and successfully applied to a clinical pharmacokinetic study of mitiglinide in 10 healthy volunteers following oral administration.     

A组轮状病毒我国地方株VP6基因的序列测定及其在杆状病毒系统中的表达

通过反转录－ 聚合酶链反应（ Ｒ Ｔ－ Ｐ Ｃ Ｒ） 获得了轮状病毒地方株 Ｔ１１４ Ｖ Ｐ６ 全基因的ｃ Ｄ Ｎ Ａ 片段,将其克隆入转移载体质粒ｐ Ｖ Ｌ１３９３ 中,构建成重组质粒ｐ Ｖ Ｌ１３９３ － Ｖ Ｐ６ 。对克隆的 Ｖ Ｐ６ 基因进行序列测定,并用它和杆状病毒（ Ａｃ Ｍ Ｎ Ｐ Ｖ） 野毒株 Ｄ Ｎ Ａ 共转染 Ｓｆ９ 细胞,筛选纯化得到含 Ｖ Ｐ６ 基因插入片段的重组杆状病毒,并进行了表达重组蛋白 Ｖ Ｐ６ 的检测。测序结果显示 Ｖ Ｐ６ 基因全长１ ３５６ｂｐ ,序列分析显示与 Ｗａ 株非常接近,提示 Ｔ１１４ 为亚组Ⅱ病毒株。用高价免疫血清经 Ｗｅｓｔｅｒｎ ｂｌｏｔ 检测表达产物,结果显示,重组病毒感染细胞裂解液样品中可见大小约４５ｋ Ｄ 的特异条带;亚组Ⅱ特异性单抗检测到大小约１２０ｋ Ｄ 的条带,提示重组蛋白 Ｖ Ｐ６ 获得了表达,具有正常的抗原反应性和天然 Ｖ Ｐ６ 的三体结构。     

共聚焦镜观察凋亡巨噬细胞内pH的变化

用透射电镜观察巨噬细胞的形态学改变,结果显示,地塞米松处理８小时后,大部分巨噬细胞发生凋亡特征变化：胞突缩短、减少,胞膜完整。胞体皱缩,胞质密度增加,其中出现大量空泡。胞核染色质边聚、浓缩。另外用激光扫描共聚焦显微镜（ＡＣＡＳ５７０）和ｐＨ荧光探针ＳＮＡＲＦ┐１／ＡＭ实时检测地塞米松处理巨噬细胞胞浆ｐＨ的动态变化。加入地塞米松,多数巨噬细胞胞浆马上发生快速和短期的碱化。随后,胞浆ｐＨ缓慢降低,胞浆酸化。结果表明,胞浆酸化是细胞凋亡发展的必然过程,胞浆碱化则很可能与细胞凋亡的发生相关,也可能与细胞种类、细胞功能状态相关     

Squamous cell carcinomas of the skin at ear tag sites in aged FVB/N mice

We report the development of squamous cell carcinomas (SCCs) of the skin at or near the site of ear tags composed of a nickel-copper alloy and used for identification during the course of a long-term study of incipient congenic FVB/N mice containing the human BCL6 transgene (FVB.Cg-Tg[tetO-BCL6]Bbn Tg[EmicroSR-tTa]83Bop), their littermate controls, and wild-type FVB/N. Of a total population of 160 mice, 14 (8.8%) developed SCCs in the tagged (right) ear after a median observation period of 25 months, but none of the animals developed tumors in the opposite ear (P = 0.0001). Nine of the fourteen mice with SCCs had to be euthanized because they were thought to be in distress from the ear condition, but the remaining five died or were euthanized for other reasons related to the research study. These animals ranged in age from 331 to 921 days at the time of death. Five of the tumors were well-differentiated (grade 1) SCCs; the remainder were grade 3 and tended to be deeply invasive neoplasms with undifferentiated areas containing a spindle cell component. One of these metastasized to kidney. When using the FVB/N mouse strain for long-term studies, it is necessary to consider that nearly 9% of the population may develop SCCs at or near ear-tag sites that may necessitate early removal of the animal.     

Cell-cycle-dependent subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse

M phase or maturation promoting factor (MPF), a kinase complex composed of the regulatory cyclin B and the catalytic p34cdc2 kinase, plays important roles in meiosis and mitosis. This study was designed to detect and compare the subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse. We found that all these proteins were concentrated in the germinal vesicle of oocytes. Shortly after germinal vesicle breakdown, all these proteins were accumulated around the condensed chromosomes. With spindle formation at metaphase I, cyclin B1 and phosphorylated cyclin B1 were localized around the condensed chromosomes and concentrated at the spindle poles, while p34cdc2 was localized in the spindle region. At the anaphase/telophase transition, phosphorylated cyclin B1 was accumulated in the midbody between the separating chromosomes/chromatids, while p34cdc2 was accumulated in the entire spindle except for the midbody region. At metaphase II, both cyclin B1 and p34cdc2 were horizontally localized in the region with the aligned chromosomes and the two poles of the spindle, while phosphorylated cyclin B1 was localized in the two poles of spindle and the chromosomes. We could not detect a particular distribution of cyclin B1 in fertilized eggs when the pronuclei were initially formed, but in late pronuclei cyclin B1 was accumulated in the pronuclei. p34cdc2 and phosphorylated cyclin B1 were always concentrated in one pronucleus after parthenogenetic activation or in two pronuclei after fertilization. At metaphase of 1-cell embryos, cyclin B1 was accumulated around the condensed chromosomes. Cyclin B1 was accumulated in the nucleus of late 2-cell embryos but not in early 2-cell embryos. Furthermore, we also detected the accumulation of p34cdc2 in the nucleus of 2- and 4-cell embryos. All these results show that cyclin B1, phosphorylated cyclin B1 and p34cdc2 have similar distributions at some stages but different localizations at other stages during oocyte meiotic maturation and fertilization, suggesting that they may play a common role in some events but different roles in other events during oocyte maturation and fertilization.