Human Pluripotent Stem Cells Have a Novel Mismatch Repair-dependent Damage Response

Human pluripotent stem cells (PSCs) are presumed to have robust DNA repair pathways to ensure genome stability. PSCs likely need to protect against mutations that would otherwise be propagated throughout all tissues of the developing embryo. How these cells respond to genotoxic stress has only recently begun to be investigated. Although PSCs appear to respond to certain forms of damage more efficiently than somatic cells, some DNA damage response pathways such as the replication stress response may be lacking. Not all DNA repair pathways, including the DNA mismatch repair (MMR) pathway, have been well characterized in PSCs to date. MMR maintains genomic stability by repairing DNA polymerase errors. MMR is also involved in the induction of cell cycle arrest and apoptosis in response to certain exogenous DNA-damaging agents. Here, we examined MMR function in PSCs. We have demonstrated that PSCs contain a robust MMR pathway and are highly sensitive to DNA alkylation damage in an MMR-dependent manner. Interestingly, the nature of this alkylation response differs from that previously reported in somatic cell types. In somatic cells, a permanent G₂/M cell cycle arrest is induced in the second cell cycle after DNA damage. The PSCs, however, directly undergo apoptosis in the first cell cycle. This response reveals that PSCs rely on apoptotic cell death as an important defense to avoid mutation accumulation. Our results also suggest an alternative molecular mechanism by which the MMR pathway can induce a response to DNA damage that may have implications for tumorigenesis.     

大剂量~(60)Co照射后造血干细胞潜在的残留损伤

本文对受到临界全致死剂量——8.5Gy~(60)Co照射后的小鼠造血干细胞(HSC)的自我更新力进行了测试,并对照射后4个月,造血功能业已恢复的小鼠HSC的造血重建功能进行了研究。结果表明:应用骨髓连续移植实验所测得的照后3个月中骨髓CFU_s的自我更新潜能明显衰退了。用照射后造血恢复小鼠的CFU_s给全致死剂量照射的受体进行移植治疗,发现其移植效力比正常的显著减弱。在受体存活30天时,CFU_s的再生速率只有正常的1/17。通过对性染色体追踪观察的资料分析,此种小鼠的??造血细胞在受体中难以形成长期稳定的嵌合体。以上事实反映出,照射后小鼠的HSC的造血重建功能大大削弱了,揭示出残存干细胞质量上的缺陷。对于这种潜在的残留损伤的机制,从“干细胞增殖力耗竭学说”和“基因自我修整假说”的角度进行了讨论。     

Application of good laboratory practice (GLP) to culture collections of microbial and cell cultures

Although the principles and the necessity for good laboratory practice (GLP) guidelines to confirm the credibility, integrity, and quality of non-clinical laboratory studies have been known for more than a decade, culture collection activities are not subject to them. Because of recent advances in biotechnology, culture collections face increased demands not only for quality cultures but also current information. When applied in culture collections, GLP guidelines prove to be an excellent management tool as well as a cost-effective system of providing authentic and reliable microbial and cell cultures and associated data.     

A Membrane-delimited Effect of Internal pH on the K+ Outward Rectifier of Vicia Faba Guard Cells

ABA stimulation of outward K⁺ current (I _K,out) in Vicia faba guard cells has been correlated with a rise in cytosolic pH (pH _i ). However, the underlying mechanism by which I _K,out is affected by pH _i has remained unknown. Here, we demonstrate that pH _i regulates outward K⁺ current in isolated membrane patches from Vicia faba guard cells. The stimulatory effect of alkalinizing pH _i was voltage insensitive and independent of the two free calcium levels tested, 50 nm and 1 μm. The single-channel conductance was only slightly affected by pH _i . Based on single-channel measurements, the kinetics of time-activated whole-cell current, and the analysis of current noise in whole-cell recordings, we conclude that alkaline pH _i enhances the magnitude of I _K,out by increasing the number of channels available for activation. The fact that the pH _i effect is seen in excised patches indicates that signal transduction pathways involved in the regulation of I _K,out by pH _i , and by implication, components of hormonal signal transduction pathways that are downstream of pH _i , are membrane-delimited. Received: 5 June 1996/Revised: 1 August 1996     

Reduced glutathione esters—antidotes to toxicity. Cytotoxicity induced by hydrogen peroxide, 1-chloro-2,4-dinitrobenzene,and menadione in murine P388D1 macrophages in vitro

Repletion of depleted cellular reduced glutathione (GSH) levels in oxidative stress and exposure to arylating agents is a strategy for the development of antidotes to chemical toxicity. The effect of GSH, reduced glutathione ethyl monoester (GSHEt), and reduced glutathione ethyl diester (GSHEt₂) on the cytotoxicity of hydrogen peroxide, 1-chloro-2,4-dinitrobenzene (CDNB), and menadione to P388D₁ macrophages in vitro was investigated. The median toxic concentration TC₅₀ values of the toxicants were hydrogen peroxide 24 ± 2 mM (N = 19), CDNB 63 ± 6 μM (N = 18), and menadione 30 ± 4 μM (N = 22). Reduced glutathione, GSHEt, and GSHEt₂ were poor antidotes to hydrogen peroxide toxicity. Indeed, the observed antidote effects were attributed to the nonenzymatic reaction of the GSH derivatives with hydrogen peroxide in the extracellular medium. Reduced glutathione ethyl diester was a more potent antidote of CDNB- and menadione-mediated toxicity than GSHEt and GSH. For cell incubations with the approximate median toxic concentration TC₅₀ values of hydrogen peroxide, CDNB, and menadione, the respective median effective antidote concentration EC₅₀ values were GSHEt 23.8 ± 4.1 mM (N = 9), 3.6 ± 0.6 mM (N = 11), and 226 ± 93 μM (N = 12); and GSHEt₂ 20.4 ± 1.9 mM (N = 6), 603 ± 2 μM (N = 9), and 7.6 ± 2.3 μM (N = 12). Reduced glutathione ethyl diester was a potent antidote to CDNB- and menadione-induced toxicities but not to hydrogen peroxide-induced toxicity under acute intoxication conditions. © 1996 John Wiley & Sons, Inc.     

Metabolic rates of vascular endothelial cellsin vitro

Cultures of endothelial cells and cell lines of endothelial origin were maintained at confluence without medium exchange for a period of 72 h. During this time period the concentration of nutrients — amino acids and glucose — and metabolic waste products — lactate and ammonium — was determined as well as cell vitality and cell numbers. Metabolic rates were calculated and compared for the different cell lines. Surprisingly the primary cells showed significantly higher rates of glucose and glutamine consumption, respectively lactate production than the immortalized cell lines. Except for one tumorigenic cell line all cells showed a significant participation of transaminases in glutamine/ammonium metabolism. Furthermore it could be shown that in routine culture there was no depletion of nutrients or critical accumulation of ammonium or lactate over a culture period of 72 h.Abbreviations BAEC bovine aorta endothelial cells - EC vascular endothelial cells - FGF fibroblast growth factor - HUVEC vascular endothelial cells from human umbilical cord veins - IF 1:1 mixture of Iscove's MDM and Ham's F12 basal media - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid - NCS newborn calf serum - PBS phosphate buffered saline - TE 0.05% (w/V) trypsin, 0.02% (w/v) EDTA in PBS     

巨噬细胞对正常人极低密度脂蛋白亚组分代谢的研究

本文研究了小鼠腹腔巨噬细胞对正常人极低密度脂蛋白(N-VLDL)两种亚组分VLDL_1和VLDL_3的代谢。两种亚组分都能以受体方式和非特异性方式被巨噬细胞摄取和降解。在受体途径中以VLDL_1的摄入量居多。对胞内甘油三酯(TG)的堆积作用以VLDL_1较强,对胆固醇酶(CE)的堆积则以VLDL_3较强。表明两者在促进巨噬细胞向泡沫细胞转变中的作用有所不同。     

利用IL－3／GM－CSF融合蛋白（PIXY－321）扩增脐血造血细胞方法的初步建立

利用单克隆抗体免疫磁珠吸附方法分离脐血ＣＤ３４＋细胞,并观察了ＩＬ３／ＧＭＣＳＦ融合蛋白（ＰＩＸＹ３２１）对脐血ＣＤ３４＋细胞的刺激作用。ＰＩＸＹ３２１对脐血ＣＤ３４＋细胞扩增作用大于ＩＬ３和ＧＭＣＳＦ单独及联合应用。在液体培养条件下,每毫升２０ｎｇＰＩＸＹ３２１可有效地扩增脐血造血祖细胞,适宜扩增时间为５－８天,扩增后造血祖细胞的数量可达扩增前的８－１０倍,从而初步建立了一种简单可行的脐血造血细胞扩增方法。     

鸡胚血液中原始生殖细胞的分离及其培养的研究

从孵化４８～５５小时鸡胚中抽取血液,每只胚胎可获血液２～６μｌ左右。一步法离心分离原始生殖细胞,可使其浓度由０．１％以下提高到５０％以上。将多余血细胞用微量吸管移走后,加入添加１０％胎牛血清的ＴＣＭ—１９９做为培养基,３７．５℃,５％ＣＯ２,９５％空气,饱和湿度下培养,原始生殖细胞可成活２４小时左右。     

Relationship between immunoglobulin levels and extremes of solar activity

The possible relationship between epidemics and extremes of solar activity has been discussed previously. The purpose of the present study was to verify whether differences in the levels of immunoglobulins (IgA, IgG, IgM) could be noted at the highest (July 1989) and lowest (September 1986) points of the last (21st) and present (22nd) 11-year solar cycle. The work was divided into a 1-month study (covering the month of minimal or maximal solar activity), a 3-month study (1 month before and after the month of minimal or maximal solar activity) and a 5-month study (2 months before and after the month of minimal or maximal solar activity). A trend of a drop-off for all three immunoglobulins was seen on the far side of the maximal point of the solar cycle. Statistical significance was achieved in the 5-month study for IgM (P=0.04), and a strong trend was shown for IgG (P=0.07). Differences between the sexes were also noted.